Hydrodynamic method and apparatus for delivering fluids to kidney tissues

ABSTRACT

The invention provides a catheter and control box apparatus and related methods that are useful in delivering liquids, including liquids comprising nucleic acid molecules into cells. In particular, the invention provides a catheter that stabilizes fluid flow within a vein to deliver a volume, pressure charge of saline solution, exogenous compositions, and isolated vectors to kidney cells, using the renal vein as a guide and under hydrodynamic pressure. The catheter and control box apparatus and related methods described herein are useful to research, prognose, ameliorate symptoms of kidney injury, and treat kidney pathologies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 61/872,230, filed Aug. 30, 2013.

BACKGROUND OF THE INVENTION

Reliable methods for gene transfer to specific target cells in liveanimals have the potential both to enhance basic and disease-focusedresearch in animal models and to facilitate the advancement of genetherapy in humans. Numerous methods have been proposed to deliverexogenous genes to mammalian cells in situ. These techniques couldprovide inexpensive and rapid alternatives to pronuclearmicroinjection-derived transgenic models. However, more efficientapproaches are needed to enhance gene transfer by improving thedistribution, extent and duration of gene expression, while minimizinginjury associated with the delivery.

Generally, in vivo nucleic acid molecule transfer rates are directlyinfluenced by the following phenomena: 1) time taken for cells toexpress the delivered nucleic acid molecules; 2) number of cells thatincorporate the exogenous nucleic acid molecules; 3) intensity of theresulting expression; 4) cellular turnover rates; 5) vascular flowrates; 6) reliability of the process; 7) method driving nucleic acidmolecule expression; and 8) possible injury that may result from thenucleic acid molecule delivery process.

Efficient gene transfer has been difficult to achieve routinely in thekidney, as illustrated by the varied levels of successful transgeneincorporation reported in previous studies, and more generally, thefailure of any of these methods to achieve widespread use. The structureof various renal vascular beds and their permeability characteristicspresent intrinsic challenges to gene transfer processes. For example,proximal tubule epithelial cells have an immense capacity for the apicalendocytic uptake of exogenous materials, and thus the possibility oftransgene incorporation. Yet, the accessibility of the apical domain toexogenously delivered vectors, and accordingly the resulting extent oftransgene uptake, are strongly limited by the permeabilitycharacteristics of the glomerular filtration barrier. The degree towhich proximal tubule cells are accessible for gene delivery at thebasolateral surface, via the peritubular capillaries, is largelyunknown.

In the kidney, previous studies have observed widely varying levels ofgene expression using adenovirus vectors. In those studies, theadenoviral vectors were delivered through arterial injections in normaland cystic rats; via pelvic catheter infusion in normal rats; and viatail vein and cortical micropuncture injections in uninjured animals.For instance, adenovirus vectors delivered through intra-arterialinjections to kidneys that were pre-chilled for extended periodsgenerated transgene expression largely within the cortical vasculature;whereas the pre-chilling treatment, combined with vasodilators,facilitated gene transfer in both the inner and outer stripes of theouter medulla. However, expression in the cystic kidneys was onlyobserved as patchy patterns in the vasculature, some epithelial cystsand interstitial cells.

Another group used adenovirus vectors to transduce rat glomerularendothelial cells by slow infusion into the renal artery. This techniqueresulted in transgene expression which lasted for at least 3 weekswithout causing significant damage. However, expression was not observedwithin other cell types. Within the same study, analogous concentrationsof the same adenovirus vector were delivered to the kidney via arterialinjections and pelvic catheter infusions produced transgene expressionin distinct, but still limited, regions of the kidney.

Comparably, studies using tail vein or retrograde ureteral adenovirusinfusions, to target aquaporin water channels, also reported variedlevels of expression that appeared to be dependent upon the transgeneinfusion site. Aquaporin 1 (AQP1) expression in apical and basolateralmembranes of proximal tubule epithelial cells in the renal cortex, butno AQP1 expression was observed in glomeruli, loop of Henle, orcollecting duct, when the virus was delivered by tail vein infusions.

Conversely, through ureteral infusions, significant ureteral and renalpapilla transgene expression was reported, also with less intense andpatchy expression observed in cortical collecting ducts.

Finally, others have explored direct transfer of adenovirus vectors intoindividual nephron segments using micropuncture techniques and achievedsite-specific genetic incorporation within the injected tubules orvascular welling points. One limitation of the approach, however, isthat gene expression is restricted to the injection site. There is alsoa risk of injury from transgene delivery via inflammatory responsesgenerated from large concentrations of adenovirus vectors. Importantly,this result also demonstrated the utility of intravital fluorescenttwo-photon microscopy as a means of directly monitoring proteinexpression in live animals.

Lastly, acute kidney injury (AKI) remains a major clinical problem, asapproximately 25% of ICU patients and 5-15% of all hospitalized patientsexperience this injury. Such patients observe increased risks of havingtheir AKI progress to renal insufficiency, and ultimately dying duringtheir hospitalization. Generally, AKI results primarily from directrenal trauma or blood loss, and the accumulation of various toxins, suchas broad-spectrum antibiotics and chemotherapeutic agents, in proximaltubule epithelial cells. The management of AKI depends on theidentification and treatment of its underlying cause, and currenttreatment regimes are mainly supportive. Intravenous fluid delivery isgenerally the first course of treatment for pre-renal AKI, in theabsence of hypervolemia. This standard approach is employed to preventor eliminate volume depletions, remove tubular blockages, dilute toxinconcentrations, facilitate diuresis and reinstate normal GFP levels.However, further studies are needed to determine exact fluid quantitiesand infusion endpoints for maximal interventional benefit.

AKI patients also have increased risk of progression to renal failure.AKI results from various etiologies including nephrotoxic agents, suchas aminoglycosides, chemotherapeutic drugs and radiocontrast dyes.Management of AKI depends on identification and treatment of underlyingcauses, and current treatment regimens are mainly supportive. Genetherapy has been proposed as a novel alternative to treat, and possiblyprevent AKI. While significant challenges to efficient renal genetransfer remain, the development of renal gene therapy by hydrodynamicgene delivery has shown promise in addressing this problem by providingsubstantial levels of reporter transgene expression in proximal tubule,which is the site of major damage during AKI.

SUMMARY OF THE INVENTION

The invention provides, inter alia, an augmented hydrodynamic method fordelivering fluid into a kidney cell of a mammalian subject, comprising:administering fluid into at least one kidney of a mammalian subjectusing the subject's renal vein as a guide for administering the fluid tothe kidney, and wherein the fluid is administered to the kidney via therenal vein, under retrograde hydrodynamic pressure, and with temporaryrenal blood vessel occlusion.

Also provided are such methods, wherein the fluid further comprises atleast one isolated nucleic acid molecule.

Also provided are such methods, wherein the isolated nucleic acidmolecule is selected from the group consisting of: plasmid; nakedplasmid; plasmids mixed with chemical carriers (polyamine); plasmidmixed with microspheres; nucleic acid in solution; virus particle;virus; combination of plasmid and virus particle; and artificialchromosome.

Also provided are such methods, wherein administration of the at leastone nucleic acid molecule has a result selected from the groupconsisting of: nucleic acid molecule delivery to renal cortex and/ormedulla; nucleic acid molecule delivery to glomerular, tubular, and/orvascular kidney cells; nucleic acid molecule expression in at least onekidney cell; increased degree of nucleic acid molecule expression in atleast one kidney cell; sustained tissue morphology changes in at leastone kidney cell; limited injury to kidney after administration of the atleast one nucleic acid molecule; increased vector passage; increasedvector efficiency; increased nucleic acid molecule and/or expressedprotein diffusion; increased types of renal cells affected by nucleicacid molecule delivery; increased cavitation of renal cells; increasedcell permeability; increased nucleic acid molecule delivery rate;increased stability of nucleic acid molecules administered; and diffusecytosolic expression of nucleic acid molecules throughout cells.

Also provided are such methods, wherein the mammalian subject isselected from the group consisting of: laboratory animal; companionanimal; draft animal; meat animal; and human.

Also provided are such methods, wherein the subject is a mammal selectedfrom the group consisting of: cat; dog; horse; bovine; and human.

Also provided are such methods, wherein the mammalian subject has akidney disease selected from the group consisting of: acute kidneyfailure; acute phosphate nephropathy; acute tubular necrosis; Alportsyndrome; amyloidosis; analgesic nephropathy; antiphospholipid syndrome;apol1 mutations; Bartter syndrome; cholesterol emboli; contrastnephropathy; cryoglobuinemia; diabetes and diabetic kidney disease;diabetes insipidus; edema, swelling; Fabry's disease; fibrillaryglomerulonephritis and immunotactoid glomerulopathy; focal segmentalglomerulosclerosis, focal sclerosis, focal glomerulosclerosis;gestational hypertension; Gitelman syndrome; glomerular diseases;Goodpasture syndrome; hematuria (blood in urine); hemolytic uremicsyndrome; high blood pressure and kidney disease; hyperaldosteronism;hypercalcemia (high blood calcium); hyponatremia (low blood sodium);hyperoxaluria; IgA nephropathy; IgG4 nephropathy; interstitial cystitis,painful bladder syndrome; interstitial nephritis; kidney stones; lightchain deposition disease, monoclonal immunoglobulin deposition disease;Liddle syndrome; loin pain hematuria; lupus, systemic lupuserythematosis; lupus kidney disease, lupus nephritis; malignanthypertension; medullary cystic kidney disease; medullary sponge kidney;membranoproliferative glomerulonephritis; membranous nephropathy;metabolic acidosis; microscopic polyangiitis; minimal change disease;multiple myeloma; nail-patella syndrome; nephrocalcinosis; nephroticsyndrome; nutcracker syndrome; orthostatic hypotension; orthostaticproteinuria; post-infectious glomerulonephritis, post-streptococcalglomerulonephritis; polycystic kidney disease; preeclampsia; proteinuria(protein in urine); pyelonephritis (kidney infection); rapidlyprogressive glomerulonephritis; renal artery stenosis; renal infarction;renal tubular acidosis; reflux nephropathy; retroperitoneal fibrosis;rhabdomyolysis; sarcoidosis; scleroderma renal crisis; thin basementmembrane disease, benign familial hematuria; tuberous sclerosis; tumorlysis syndrome; urinary tract infection; urinary tract obstruction; vonHippel-Lindau disease; warfarin-related nephropathy; and Wegener'sgranulomatosis.

Also provided are such methods, which further comprise a step prior toadministering the fluid into the renal vein of a mammalian subject, theprior step selected from the group consisting of: administering anadjuvant; administering an anesthetic; administering an anticoagulant;administering a contractile agent; administering a relaxant agent; andadministering a blood volume agent.

Also provided are such methods, which further comprises monitoringnucleic acid molecule delivery.

Also provided are such methods, wherein monitoring is accomplished by amethod selected from the group consisting of: intravital multiphotonfluorescence microscopy and confocal laser scanning microscopy.Alternatively, or in addition to, for clinical purposes, monitoring maybe provided from the group consisting of: pet scanning, Doppler flowultrasound, MRI with contrast, CT scan with contrast, angiograms of thekidney.

The present invention also provides methods for delivering at least onenucleic acid molecule to kidney cell of a mammalian subject, comprising:injecting a vector comprising at least one nucleic acid molecule intothe mammalian kidney of a subject using the renal vein as a guide andunder retrograde pressure.

Also provided are such methods, which further comprises clamping a bloodvessel in the kidney so as to augment delivery of the nucleic acidmolecule to the subject.

Also provided are such methods, wherein the vector is a viral vector.

Also provided are such methods, wherein the vector comprises humankidney regulatory elements.

Also provided are such methods, wherein the vector comprises a nucleicacid molecule useful to treat or prevent a kidney disease or condition.

Also provided are such methods to treat a kidney pathology in a subjecthaving a kidney pathology, comprising: administering an appropriatelytherapeutic fluid according to a method herein to a subject having akidney pathology and treating a kidney pathology in the subject.

Also provided are such methods to prevent a kidney pathology in asubject at risk of kidney pathology, comprising: administering anappropriately therapeutic fluid according to a method herein to asubject having a kidney pathology and preventing a kidney pathology inthe subject.

Also provided are such methods to ameliorate at least one symptomrelated to a kidney pathology in a subject, comprising: administering anappropriately therapeutic fluid according to a method herein to asubject having a kidney pathology and ameliorating at least one symptomrelated to a kidney pathology in the subject.

Also provided are such methods to ameliorate at least one symptomrelated to acute kidney injury in a subject with a symptom related toacute kidney injury, comprising: administering an appropriatelytherapeutic fluid according to a method herein to a subject having acutekidney injury and ameliorating at least one symptom related to acutekidney injury in the subject.

Also provided are such methods, wherein the fluid comprises salinesolution.

Also provided are such methods to prevent or ameliorate at least onesymptom related to ischemia/reperfusion kidney injury in a subject atrisk of, or having, a symptom related to ischemia/reperfusion kidneyinjury, comprising administering an appropriately therapeutic fluidaccording to a method herein to a subject at risk of, or havingischemia/reperfusion kidney injury and preventing or ameliorating atleast one symptom related to ischemia/reperfusion kidney injury in thesubject.

Also provided are such methods wherein the fluid comprises salinesolution and/or at least one exogenous nucleic acid.

The present invention also provides methods to introduce at least oneexogenous nucleic acid into at least one kidney cell of a subject inneed thereof, comprising administering a fluid comprising at least oneexogenous nucleic acid via retrograde hydrodynamic delivery of the fluidvia the renal vein to at least one kidney cell of a patient in need ofsuch administration, and wherein administration also includes temporaryrenal blood vessel occlusion, thereby introducing at least one exogenousnucleic acid into at least one kidney cell of a patient in need thereof.

Also provided are such methods wherein the length of time the fluid isadministered is selected from the group consisting of: approximately 1second to approximately 60 seconds; approximately 1 second toapproximately 50 seconds; approximately 1 second to approximately 40seconds; approximately 1 second to approximately 30 seconds;approximately 1 second to approximately 20 seconds; approximately 1second to approximately 10 seconds; approximately 1 second toapproximately five seconds; approximately five seconds.

Also provided are such methods wherein one or more exogenous nucleicacids are introduced at an efficiency selected from the group consistingof: approximately 10% or greater; approximately 20% or greater;approximately 30% or greater; approximately 40% or greater;approximately 50% or greater; approximately 60% or greater;approximately 70% or greater; approximately 80% or greater;approximately 90% or greater.

Also provided are such methods wherein one or more exogenous nucleicacids are introduced at an efficiency selected from the group consistingof: greater than 50%; 40% to 86%; and 78% to 86%.

Also provided are such methods wherein one or more exogenous nucleicacids are introduced into at least one superficial cortex cell at anefficiency selected from the group consisting of: approximately greaterthan 70%; approximately greater than 80%, and approximately greater than90%.

Also provided are such methods wherein one or more exogenous nucleicacids are introduced at a depth of at least 100 μm and at an efficiencyselected from the group consisting of: approximately 40% or greater;approximately 50% or greater; approximately 60% or greater;approximately 70% or greater; approximately 80% or greater; andapproximately 90% or greater.

Also provided are such methods wherein at least some exogenous nucleicacids are retained in the at least one kidney cell for a time periodselected from the group consisting of: greater than 2 days; greater than3 days; greater than 4 days; greater than 5 days; greater than 6 days;greater than 7 days; greater than 14 days; greater than 21 days; andgreater than 28 days.

Also provided are such methods wherein the exogenous nucleic acids areintroduced to a depth of kidney cells selected from the group consistingof: at least about 100 μm; at least about 200 μm; at least about 300 μm;at least about 400 μm; at least about 500 μm, and greater than 500 μm.

Also provided are such methods, wherein the exogenous nucleic acids areintroduced to kidney cells in a structure selected from the groupconsisting of: superficial cortex; cortex; cortico-medullary junction;medulla; nephron; glomerulus; cortical and medullary collecting duct;and distal tubules.

Also provided are such methods, wherein the exogenous nucleic acids areintroduced to kidney selected from the group consisting of: apicalmembranes; basolateral membranes; tubular epithelial cells; glomularcells; nephron cells; tubular interstitial cells; endothelial cells;fibroblasts; pericytes endogenous stem cells; and tubular lumen cells.

Also provided are such methods, wherein efficiency is estimated by ameasurement selected from the group consisting of: renal cell uptake;expression of at least one exogenous nucleic acid; at least onebiomarker alteration; at least one chemical marker alteration; at leastone cellular marker alteration; at least one structural markeralteration; at least one functional marker alteration; at least one cellviability marker alteration; at least one cell metabolism markeralteration; and at least one cell morphology marker alteration, whereinany alteration is measured compared to pre-administration of exogenousnucleic acid.

Also provided are such methods, wherein the at least one exogenousnucleic acid is a gene.

Also provided are such methods, wherein the at least one exogeneousnucleic acid is administered via an adenovirus.

Also provided are such methods, wherein the at least one exogenousnucleic acid is administered via a plasmid.

Also provided are such methods, wherein the nucleic acid is selectedfrom the group consisting of: isocitrate dehydrogenase 2; andsulphotransferase.

Also provided: gene therapy using any of the above compositions ormethods; drug discovery using any of the above compositions or methods;kits using any of the above compositions or methods; assays using any ofthe above compositions or methods; compositions comprising any of theabove compositions or methods; formulations comprising any of the abovecompositions or methods and using any of the above compositions ormethods.

The terms “treat”, “treatment,” and “treating” and/or “ameliorating”include pathology reduction, reduction in symptoms, preventative (e.g.,prophylactic) and palliative care.

In addition to the augmented hydrodynamic method for delivering fluidinto a kidney cell of a mammalian subject, disclosed above, theinvention includes embodiments of a hydrodynamic pressure deliverycatheter, a hydrodynamic pressure delivery system, and a method ofproviding a hydrodynamic pressurized fluid charge to an organ, such as akidney, as illustrated in the following examples. The variousembodiments of the invention may comprise, individually and/or incombination, one or more of the following features from these examples:

Example 1

A hydrodynamic pressure delivery catheter may comprise an injectionlumen terminating in an insertion end and a stabilizer, the stabilizerconfigured to dampen vibratory responses of the insertion end during afluid delivery event.

Example 2

The hydrodynamic pressure delivery catheter of example 1 wherein thestabilizer is a fluid inflatable element having a plurality of radiallyexpanding segments, the radially expanding segments configured to engagea tissue lumen wall segment when deployed.

Example 3

The hydrodynamic pressure delivery catheter of example 2 wherein theplurality of radially expanding segments are at least three radiallyexpanding segments that are arranged in a generally equal spacing arounda circumference of the catheter.

Example 4

The hydrodynamic pressure delivery catheter of example 2 wherein theplurality of radially expanding segments are also configured to rotateduring deployment of the segments.

Example 5

The hydrodynamic pressure delivery catheter of example 3 wherein theradially expanding elements include a thickened section configured tocontact a tissue wall when deployed and a thinned section configured toexpand circumferentially when the radially expanding elements areinflated.

Example 6

The hydrodynamic pressure delivery catheter of example 1 wherein thestabilizer is a single, radially expanding element that permits at leasta portion of a blocked fluid to pass.

Example 7

The hydrodynamic pressure delivery catheter of example 6 wherein thesingle, radially expanding element is one of a spiral expanding element.

Example 8

The hydrodynamic pressure delivery catheter of example 2 wherein anoccluding balloon is configured to prevent fluid flow when radiallyexpanded, the occluding balloon being spaced apart from the stabilizer.

Example 9

The hydrodynamic pressure delivery catheter of example 9 wherein a trapsection is spaced between the occluding balloon and the stabilizer, thetrap section being configured to secure a portion of the tissue lumenwall section such that the catheter is secured within a tissue lumenwhen the occluding balloon and the stabilizer are radially expanded.

Example 10

The hydrodynamic pressure delivery catheter of example 9 wherein apressure sensor is positioned proximate to the injection lumen andconfigured to provide a feedback signal to regulate fluid flow throughthe injection lumen.

Example 11

The hydrodynamic pressure delivery catheter of example 10 wherein a pumpis in fluid communication with the injection lumen, the stabilizer, andthe occluding balloon, the pump having a controller and a controlalgorithm that are responsive to the feedback signal to regulate fluidpressure in at least one of the occluding balloon, the stabilizer, andthe injection lumen.

Example 12

A hydrodynamic pressure delivery system comprising: a catheter having aninjection lumen and a stabilizer; and a pump having a fluid containmentvessel configured to deliver a pressurized fluid charge through theinjection lumen, the pressurized fluid charge having a vibrationexcitation force component that is substantially counteracted by thestabilizer, the pump further including a controller that is configuredto maintain the pressurized fluid charge at an efficacious deliveredvolume over a predetermined time sufficient to cause fluid uptake in atarget tissue cell.

Example 13

The hydrodynamic pressure delivery system of example 12 wherein thefluid containment vessel is a syringe, and the controller includes acontrol algorithm configured to control the pressurized fluid chargesuch that the pressurized fluid charge is an about 60 ml fluid chargedispensed in about a one minute time period.

Example 14

The hydrodynamic pressure delivery system of example 13 wherein thecatheter includes at least one pressure sensor configured to providepressure data to the control algorithm such that the control algorithmadjusts the fluid charge delivery rate to maintain the efficaciousdelivered fluid volume over the predetermined time in response to apressure signal from the at least on pressure sensor.

Example 15

The hydrodynamic pressure delivery system of example 12 wherein theefficacious delivered fluid volume over a predetermined time is in arange of about 0.75 to about 1.25 ml per second.

Example 16

The hydrodynamic pressure delivery system of example 13 wherein thecontrol algorithm is configured to control at least one of an occludingballoon pressure and a stabilizer pressure.

Example 17

The hydrodynamic pressure delivery system of example 16 wherein theoccluding balloon is in fluid communication with an occluding pump andthe stabilizer is in fluid communication with a stabilizer pump, theoccluding pump and stabilizer pump configured to sense and adjust thepressure of the occluding balloon and stabilizer such that blood flow isoccluded and the injection lumen is stabilized in response to forcescreated by the pressurized fluid charge during a fluid delivery event.

Example 18

A method of providing a hydrodynamic pressurized fluid charge to akidney, the method comprising the steps of: a) providing a fluidcontainment vessel having a fluid configured for delivery to the kidney;b) inserting a catheter having a stabilizer and an injection lumen intoone of a renal vein and a renal artery; c) occluding blood flow throughthe one of the renal vein and artery; and d) delivering a fluid chargeat a rate of about 1 ml per second.

Example 19

The method of example 18 wherein the fluid is selected from a group ofone of nucleic acids, adenovirus vectors, stem cells, renal epithelialcells, fibroblasts, endothelial cells, plasmids, artificial chromosomes,retroviruses, adenovirus, adeno-associated virus, anti-sense DNA, siRNA,ShRNA, RNAi, Organelles-mitochondria, peroxisomes, endosomes, exosomesHormones, growth factors, peptides, derivatized peptides and proteins,glycosylated proteins, non-glycosylated proteins sugars, sugar,polymers, drugs, saline, lactated ringers, saline with glucose, andbicarbonate.

Example 20

The method of example 18 wherein the step of delivering a fluid chargeincludes delivering 60 ml of fluid in a one minute time period to ahuman kidney.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to theaccompanying drawings forming a part of this specification wherein likereference characters designate corresponding parts in the several views.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (A) Schematic illustration of the hydrodynamic injectionprocedure. Following laparotomy to expose the left kidney, both therenal artery (red) and vein (blue) are clamped. Reagents to be deliveredare injected into the renal vein at a site between the clamp and thekidney. (B) Pressure measured in the renal vein during the hydrodynamicdelivery procedure. Pressures were measured using a damped ultrasonicDoppler flowmeter attached to a catheter inserted into the renal veinbetween the clamp and the kidney. P1: after both vascular clamps wereapplied; P2: hydrodynamic injection; P3: clamps removed. (C) Schematicillustration of the method used to analyze the efficiency oftransfection in different regions of the kidney. The figure shows amontage of Texas Red-phalloidin labeled sections collected with a 60×objective and covering a wedge of the kidney extending from cortex tohilum. Efficiency of transfection was estimated in 100×1000 μm stripeslocated at various distances from the cortical surface as illustrated.(D)-(I) Organs (kidney; D, E, G, H), lung (LU), liver (LV), heart (HR)and spleen (SP) (F & I)) recovered from animals following hydrodynamicdelivery of Toluidine Blue dye with (D-F) or without (G-I) clamping therenal artery and vein. The left kidney was injected in all cases.

FIG. 2. Intravital imaging shows expression of fluorescent proteins fromplasmid vectors. (A, D, G). Rat kidneys prior to hydrodynamic injection.Characteristic autofluorescence signal is detected in both the red andgreen channels. (B, C, E, F, H, I) Two representative fields collectedfrom the same animals as in (A, D or G), using the same imagingparameters, 3 days after injection of saline (B, C), EGFP plasmid (E, F)or EGFP-tubulin plasmid (H, I). Arrowheads indicate tubular epithelialcells expressing the fluorescent proteins. (J). 3D rendering of a volumecollected from an animal 3 days after injection of EGFP-occludin plasmid(green). Nuclei are labeled with Hoechst (blue). (K, L). A rat 1 dayafter injection of plasmid encoding tdTomato-histone H2B (red). Nucleiin (L) are labeled with Hoechst (blue). DT: distal tubule; PT: proximaltubule. Bars in all panels are 60 μm.

FIG. 3. Time course of expression of EGFP-actin from plasmid vectors.(A, D, G). Autofluorescence prior to injection. (B, C, E, F, H, I)Representative field, at two different magnifications, 3 (B, C), 14 (E,F) and 28 days (H, I) after hydrodynamic injection. Arrowheads indicateactin fluorescence in the brush border microvilli in proximal tubules.DT: distal tubule; PT: proximal tubule. Bars are 60 μm.

FIG. 4. Expression of EGFP-actin from adenoviral vectors. (A)Autofluorescence prior to injection. (B, C, D) Images collected 3 (B), 7(C) or 14 (D) days after injection. Arrowheads show expression inproximal tubule epithelial cells. DT: distal tubule; PT: proximaltubule. Bars are 60 μm.

FIG. 5. Comparison of rats injected with EGFP-actin (B) or RFP-actin (D)adenovirus. Images were collected 3 days after injection. (A, C) Imagescollected prior to injection. DT: distal tubule; PT: proximal tubule.Bars are 60 μm.

FIG. 6. Expression of EGFP-actin (B, C, D, E) from plasmid vectors inother kidney cell types (see text). (A) Autofluorescence observed 3 daysfollowing saline injection. Expression of EGFP-actin 3 (B, D, E) or 5(C) days after injection. (F) Expression of td-Tomato-H2B (red) one dayafter injection. Nuclei are labeled with Hoechst (blue). GL: glomerulus;PT: proximal tubule; V: microvasculature; S1: S1 segment of proximaltubule; AD: adipocyte in perirenal fat; RC: renal capsular cells. Barsare 60 μm.

FIG. 7. Quantitative analysis of fluorescent protein expressionfollowing hydrodynamic delivery. (A, B) montages collected from fixedkidneys 3 days following injection of saline (A) or EGFP-tubulin (B).(C) Expression of EGFP-tubulin from plasmid vectors; expression ofEGFP-actin from baculovirus or adenoviral vectors at the indicateddistances from the cortical surface of the kidney 3 days afterinjection. (D) Expression of EGFP-actin from plasmid or adenoviralvectors estimated from intravital fields at the indicated timesfollowing injection.

FIG. 8. Assessment of kidney structure and function followinghydrodynamic injection and expression of fluorescent proteins. (A, B, C)Intravital imaging of rat kidneys ˜20-30 minutes following hydrodynamicinjection of a 150 kDa TRITC dextran (red). The dextran is rapidlyinternalized by proximal tubule epithelial cells (A), is visible at thebasolateral surface (arrowhead in (A)) and frequently detected at theapical surface of these cells (arrowheads in B). In some instances,bright fluorescence was detected in the lumen of the tubule (C). (D) Ratkidney 3 days following injection of EGFP-actin plasmid (green). Thekidney was injected with 3 kDa Cascade Blue dextran and 150 kDa TRITCdextran via the jugular vein ˜20 minutes prior to imaging. Arrowheadshows abundant endocytosis of dextran in cells that express high levelsof the fluorescent protein. (E) Rats were injected with 150 kDa FITCdextran via the jugular vein 5 minutes prior to hydrodynamic injectionof saline into the renal vein. FITC dextran is confined to thevasculature (arrowhead) and is not detected at significant levels in thetubule lumen. (F) Injection of 150 kDa FITC dextran 20 minutes followinghydrodynamic injection of saline. FITC fluorescence remains confined tothe vasculature. (G, H) H&E stained sections from kidneys 3 days aftersaline (G) or EGFP-actin (H) injection. PT: proximal tubule; V:microvasculature; L: tubule lumen; GL: glomerulus.

FIG. 9A-FIG. 9F. These data provide signs of intact renal structural andfunction capacities post hydrodynamic transgene delivery. The data aretaken from a live rat 3 days after it was treated with pEGFP andPEGFP-Actin naked plasmin vectors. Images (A-C) outline pEGFP and (D-F)outline pEGFP-Actin transgene expression in proximal tubule (PT)epithelial cells. Solutions containing 3 kDa Cascade blue and 150 kDaTRITC dextrans were infused into the jugular veins of live rats. Robustand widespread uptake of the low molecular weight dextran solutions wasobserved after dye infusion, presented in images (B) and (E). TheCascade blue dextran was rapidly filtered by glomeruli, and was thenendocytosed by into proximal tubule epithelial cells. Additionally, thelarge molecular weight dextran was restricted to the vasculature asshown in images (C) and (F), as observed in FIGS. 2A and 2B. Images (Cand F) are the merger of blue, green and red channels.

FIG. 10. A measure of the changes in venous pressure that occurthroughout a hydrodynamic injection (with vascular clamps) of 0.5 mlsolution into the left renal vein of a live rat.

FIG. 11A-FIG. 11C. Intravital multiphoton micrographs, taken with a 60×objective, from two live rats within 20 minutes of receivinghydrodynamic infusions of 0.5 ml saline containing 4 kDa FITC and 150kDa TRITC dextrans, and Hoechst 33342 in (A) a normal rat; and (B) and(C) a rat with significant renal injury (hydrodynamic injection wasgiven 1 hour after a 45 minute bilateral renal occlusion). In (A), 1.5×digital zoom, we observe intense TRITC signals confined to thevasculature, FITC dextran molecules that appear to bound brush borders(arrowhead) and as endocytosed puncta within proximal tubule (PT)epithelial cells, and accumulation of the FITC dye within the lumens ofthe distal tubules (DT). These observations provide evidence of intactstructural and functional renal capacities and widespread delivery ofexogenous materials. In comparison, the relatively lower signal from theTRITC dextran within the vasculature (V) in (B) 1.5× zoom and (C)signifies a reduction in renal blood flow, deformed and denatured nucleiwithin PTs, DTs, and the vasculature (arrows)-hallmarks of apoptosis,and reduced level of renal filtration (reduced concentration of FITCmolecules and blebs within distal tubule lumens), are characterized bysever ischemia/reperfusion injuries. Nevertheless, there is stillwidespread uptake of the exogenous materials in this injury model. Red,green and blue pseudo-colors are merged in show the presence of eachprobe. All images present a merger of signals derived from Hoechst 33342labeled nuclei (blue pseudo-color signal) tissue auto fluorescence(green pseudo-color signal) and dye-based fluorescence (red pseudo-colorsignal).

FIG. 12A-FIG. 12B. Intravital multiphoton micrographs taken: (A) beforehydrodynamic delivery (tissue autofluorescence), (B) 3 days afterhydrodynamic deliver of Actin-GFP plasmids in the same rat (1.5× opticalzoom to highlight transgene expression pattern along brush borders).Arrowheads indicate the regions of enhanced transgene-based fluorescencealong the brush border of proximal tubule (PT) epithelial cells andwithin distal tubule epithelial (PT) cells. Red and green pseudo-colorsare merged in these images to differentiate between transgene and innatetissue fluorescence signals.

FIG. 13A-FIG. 13D. Multiphoton fluorescent microscopic images taken froma live rat with mild ischemia/reperfusion injury 3 days after theinitial insult: (A) image taken from a rat that did not receive anytransgene or saline treatment. Structural damage can be seen withinproximal tubules (PT) by debris within tubules lumens; (B), (C) and (D)images taken from separate rats that were subjected to hydrodynamictransgene delivery of Actin-GFP plasmids 1 hour after a 15 minutebilateral renal clamp. Enhanced transgene-based fluorescence can be seenwithin intact proximal tubule (PT) epithelial cells (arrowheads). Again,deformed nuclei within proximal (PT) and distal tubules (DT), and thevasculature (arrowheads) are hallmarks of apoptosis, which are expectedwith this ischemia/reperfusion injury. Red and green pseudo-colors aremerged in these images to differentiate between transgene and innatetissue fluorescence signals.

FIG. 14A-FIG. 14D. Fluorescent microscopic images taken from a live ratwith moderate ischemia/reperfusion injury 3 days after the initialinsult: (A) image taken from a rat that did not receive any transgene orsaline treatment. Structural damage can be seen within proximal tubules(PT) by debris within tubule lumens; (B), (C) and (D) images taken fromseparate rats that were subjected to hydrodynamic transgene delivery ofActin-GFP plasmids 1 hour after a 45 minute bilateral renal clamp.Enhanced transgene-based fluorescence can be seen within intact proximaltubule (PT) epithelial cells and within the lumens of occluded tubules(arrowheads). In (C) Hoechst 33342 was added to label nuclei. Red andgreen pseudo-colors are merged in these images to differentiate betweentransgene and innate tissue fluorescence signals. In certain cases theinjury was so severe that is was difficult to identify specific renalsegments as seen in (D).

FIG. 15A-FIG. 15D. Fluorescent microscopic images taken from a live ratwith moderate ischemia/reperfusion injury 3 days after the initialinsult: (A) image taken from a rat that did not receive any transgene orsaline treatment. Structural damage can be seen within proximal tubules(PT) by debris within tubule lumens; (B), (C) and (D) images taken fromseparate rats that were subjected to hydrodynamic transgene delivery ofActin-GFP plasmids 24 hours after a 45 minute bilateral renal clamp.Enhanced transgene-based fluorescence can be seen within intact proximaltubule (PT) epithelial cells and within the lumens of occluded tubules(arrowheads). Again, deformed nuclei within proximal (PT) and distaltubules (DT), and the vasculature (arrows) are hallmarks of apoptosis,which are expected with this ischemia/reperfusion injury. Red and greenpseudo-colors are merged in these images to differentiate betweentransgene and innate tissue fluorescence signals.

FIG. 16. Influence of hydrodynamic isotonic fluid delivery on serumcreatine levels after ischemia-reperfusion kidney injury in rats.

FIG. 17. Hydrodynamic fluid delivery appears to have a therapeuticeffect in rats with acute ischemia/reperfusion injury.

FIG. 18. Rats Hydrodynamically treated with plasmids encodingmitochondrial proteins appear to be less susceptible to acuteischemia-reperfusion injury.

FIG. 19. An intravital multiphoton micrograph of Texas Red labeledalbumin in live rat proximal (PT) and distal (DT) tubules and thevasculature (V), approximately 20 minutes it after it hydrodynamicallydelivered through the left renal vein of a rat. This 1 ml fluorescentsolution was injected at an approximate rate of 0.1 ml/s, using a PESOcatheter that was inserted into the left renal vein. The venouscatherization resulted in vasculature constriction, reduced luminalsurface area of PT epithelial cells, and fluorescent vesicles andnon-fluorescent blebs within tubule lumens. The image, taken with a 60×water objective lens, presents a merger of signals derived from tissueauto fluorescence (green pseudo-color signal) and dye-based fluorescence(red pseudo-color signal).

FIG. 20A-FIG. 20C. Intravital multiphoton micrographs taken within 20minutes after the simultaneous infusion of low (either 3 kDa CascadeBlue or 4 kDa FITC) and large (150 kDa TRITC) dextrans. These dataillustrate the effects that result from varying the hydrodynamicinjection rate and method (lower infusion volume and added vascularclamping). Each retrograde injected was performed using a 30-gaugeneedle. Signs of intact nephron structure and function are observed inimage: (A) 10-second long hydrodynamic injections, without vascularclamps, of 1 ml solution containing 3 kDa Cascade Blue and 150 kDa TRITCdextrans, and (B) 5-second long injections (injection rate 0.1 ml/s),with vascular clamps, of 0.5 ml solution containing 4 kDa FITC and 150kDa TRITC dextrans (Hoechst was added to label nuclei). In comparison,image (C) outline that 4-minute long injections (injection rate 0.0042ml/s), without vascular clamps, of 1 ml saline containing 3 kDa CascadeBlue and 150 kDa dextrans, produce vascular constriction, tubularblockage and filtration of the large 150 kDa as observed in FIG. 1.These are the mergers of blue, green and red pseudo-colors originatingfrom the low and large molecular weight dextrans.

FIG. 21A-FIG. 21D. Live rat kidney tubules micrographs obtained fromanimals prior to and 3 days after they received sham and hydrodynamicinjections of saline: (A) rat kidney imaged prior to a sham injection,(B) kidney imaged 3 days after receiving a sham injection, (C) ratkidney imaged prior to a hydrodynamic injection of saline, (D) kidneyimaged 3 days after receiving a hydrodynamic injection of saline.

FIG. 22A-FIG. 22F. Transgene expression recorded in live Sprague Dawleyrats that received hydrodynamic injections (augmented with vascularclamps) of EGFP and EGFP-Tubulin plasmid vectors. Image (A), was takenfrom a rat prior to its treatment with pEGFP naked plasmid vectors, and(B) and (C) were taken from that animal 3 days after it was treated withpEGFP naked plasmid vectors. Similarly, image (D), was taken fromanother rat prior to its treatment with pEGFP-Tubulin naked plasmidvectors, and (E) and (F) were taken from that animal 3 days after it wastreated with pEGFP-Tubulin naked plasmid vectors. Transgene expressioncan be seen within live distal tubules (DT), image (F), and proximaltubules (PT), images (B), (C), and (E). Red and green pseudo-colors weremerged to differentiate between ECFP and autofluorescence signals.

FIG. 23A-23D. A comparison of fluorescent micrographs taken from liveSprague Dawley rats that received hydrodynamic injections of GFP-Actinand RFP-Actin adenovirus vectors: image (A) was recorded in a rat priorto transgene delivery of GFP-Actin adenovirus vectors; image (B) wastaken from that animal 3 days post delivery of GFP-Actin adenovirusvectors; image (C) was recorded prior to transgene delivery of RFP-Actinadenovirus vectors; and image (D) was taken from that animal 3 days postthe delivery of RFP-Actin adenovirus vectors. Red and greenpseudo-colors were merged to distinguish between fluorescence (GFP andRFP) and autofluorescence signals.

FIG. 24A-24F. Simultaneous transgene expression observed in MDCK cellsand Sprague Dawley rat kidneys with both GFP-Actin and RFP-Actinadenovirus vectors. The cells were imaged 1 day after incubation withthe adenovirus vectors, with the ex vivo kidney images were taken fromwithin the superficial cortex of a freshly excised whole kidney. Thekidney was harvested from a rat 3 days after it was injection of theadenovirus vectors, and was imaged within 5 minutes after its excision.Red and green pseudo-colors were merged to distinguish betweenfluorescence (GFP and RFP) and autofluorescence signals, and highlightregions with co-transgene expression.

FIG. 25A-FIG. 25D. A comparison of hydrodynamic-based transgeneexpression in live glomeruli using adenovirus and plasmid vectors invarious rats 3 and 7 days post transgene delivery: (A) image of aglomerulus taken from a kidney treated with saline (control) 3 days posthydrodynamic injection; (B) image of a glomerulus taken from a kidneytreated with GFP-Actin adenovirus vectors 7 days post hydrodynamicinjection; and (C) and (D) images of glomeruli taken from kidneystreated with EGFP-Actin plasmid vectors 3 days post hydrodynamicinjection. Prior to obtaining images (C) and (D), 150 kDa TRITC dextransolutions were infused through the jugular veins to outline theglomerular capillaries and supporting vasculature and investigatestructural and functional capacities of nephron segments after thetransgene delivery process. Red and green pseudo-colors were merged todistinguish between GFP and autofluorescence signals.

FIG. 26A-FIG. 26C. Transgene expression in observed in cells surroundingthe vasculature (A) and (B), and (C) adipose tissue of the perirenalfat. The images were taken close to the renal capsule in a rat 3 daysafter it received a hydrodynamic injection of EGFP-Actin plasmidvectors. A 150 kDa TRITC dextran solution were infused through jugularveins to outline vasculature (V). Red and green pseudo-colors weremerged to distinguish between fluorescence (GFP and RFP) andautofluorescence signals.

FIG. 27. An embodiment of a hydrodynamic pressure delivery catheterhaving a stabilizing structure, shown in a collapsed configuration priorto insertion.

FIG. 28A. The hydrodynamic pressure delivery catheter of FIG. 27, shownin a free state inflated configuration.

FIG. 28B. An end view of the hydrodynamic pressure delivery catheter ofFIG. 28A, taken along arrow 28B.

FIG. 28C. An alternative embodiment of an occluding balloon for ahydrodynamic pressure delivery catheter, similar to the catheter of FIG.28A.

FIG. 29. The hydrodynamic pressure delivery catheter of FIG. 27including fluid delivery passage ways and electronic monitoringconnections.

FIG. 30. The hydrodynamic pressure delivery catheter of FIG. 29 showingan embodiment of connection elements for fluid delivery and monitoringelements.

FIG. 31. An embodiment of a hydrodynamic pressure delivery cathetershowing an dimensional arrangement suitable for a renal hydrodynamicpressure delivery system.

FIG. 32. A pressure versus time plot, similar to FIG. 10, of anembodiment of a hydrodynamic pressure delivery method suitable for usewith various embodiments of the hydrodynamic pressure delivery catheter.

FIG. 33. The hydrodynamic pressure delivery catheter of FIG. 27, showninserted and inflated in a lumen, such as a vein or artery.

FIG. 34. An alternative embodiment of a stabilizing structure portion ofa hydrodynamic pressure delivery catheter.

FIG. 35A. Another alternative embodiment of a stabilizing structureportion of a hydrodynamic pressure delivery catheter, shown in afree-state expanded condition.

FIG. 35B. A cross sectional view of the stabilizing portion of thehydrodynamic pressure delivery catheter of FIG. 35A.

FIG. 36. A cross sectional, end view of a deployment embodiment of thestabilizing portion of FIG. 35B.

FIG. 37. A cross sectional, end view of yet another alternativeembodiment of a stabilizing structure portion of a hydrodynamic pressuredelivery catheter.

FIG. 38. A perspective view of an embodiment of a hydrodynamic pressuredelivery pump for use with a hydrodynamic pressure delivery catheter.

FIG. 39. A cross sectional, schematic view of the hydrodynamic pressuredelivery pump of FIG. 38.

FIG. 40. A cross sectional view of a human torso showing an embodimentof a surgical method using an embodiment of hydrodynamic pressuredelivery catheter similar to FIG. 33.

FIG. 41. An enlarged, close up cross sectional view the kidney of FIG.40 showing the hydrodynamic pressure delivery catheter inserted throughthe renal vein.

FIG. 42. A detailed pressure versus time plot of the plot of FIG. 32illustrating an embodiment of a hydrodynamic pressure delivery method,programmable in an algorithm that is suitable for use with variousembodiments of the hydrodynamic pressure delivery catheter.

FIG. 43. A table showing test results of achieved pressures for measuredvolumes and fluid flow rates of animal kidney trials conducted inaccordance with embodiments of the invention described herein.

Before explaining the disclosed embodiment of the present invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of the particular arrangement shown, sincethe invention is capable of other embodiments. Exemplary embodiments areillustrated in referenced figures of the drawings. It is intended thatthe embodiments and figures disclosed herein are to be consideredillustrative rather than limiting. Also, the terminology used herein isfor the purpose of description and not of limitation.

DETAILED DESCRIPTION OF THE INVENTION

The inventors designed and characterized a method that utilizes renalvein-guided, retrograde pressurized injections to elicit transgeneexpression in mammalian kidneys. The inventors injected fluorescentalbumin and dextrans into rodent renal veins under hydrodynamicpressure. These molecules were observed throughout renal segments usingintravital fluorescence multiphoton microscopy. Thereafter, nakedplasmids and baculovirus vectors, which express generalized and actin-and tubulin-targeting green fluorescent proteins, were introduced intolive rodent kidneys in a similar fashion. Gene expression was thenobserved in live and ex vivo kidney segments using intravitalmicroscopy, and confirmed in vitro with confocal laser scanningmicroscopy. The inventors recorded widespread transgene expression inlive glomerular, tubular and vascular segments beyond a month after theintroduction of the transgenes. Moreover, the naked plasmids providedtwo-fold increases in gene transfer efficiencies, with sustained tissuemorphology.

The inventors have presented a method to rapidly deliver and monitorexogenous transgenes in live mammalian kidneys. In devising thistechnique, the inventors considered the following four criteria toachieve successful transformation: 1) a viable infusion site and 2)vascular manipulations to both produce widespread transgene delivery; 3)significant vector particle uptake by several renal cell types; and 4)limited general injury and vector derived toxicity.

In so doing, the inventors first determined which type of gene deliverymethod could potentially be used to overcome the innate structuralbarriers within the kidney, and supply a variety of renal compartmentswith exogenous genetic materials. Second, focus was then directed onidentifying whether the hydrodynamic forces, generated from pressurizedinjections, would aid the passage of transgenes across epithelial andendothelial tissue structures, and ultimately their cellularincorporation. Third, it was then necessary to deduce which directinfusion port (renal artery, renal vein or ureter) would possess optimalcharacteristics (responses to contractile and relaxant agonists, andvariations in compliance relative to increased blood volume) towithstand the effects of a pressurized injection.

Specifically, an optimal injection port would allow for a timelyinduction of hemostasis and minimize ischemia-reperfusion injury. Thisin turn would ideally permit the kidney to recover in a timely manner,providing no significant injury resulted from the injection process.Finally, it was necessary to investigate whether the choice of vectorwould generate appreciable levels of transgene expression in anefficacious manner.

The initial approach considered the renal artery as the infusion port.However, this method inhibited timely hemostasis after the injection andproduced significant injury to kidney, low survival rates and rare signsof transduction. The inventors then switched to renal vein infusions.Using this injection site, the inventors considered a variety of vectorsand tissue cavitation mechanisms for transgene expression. Naked DNAplasmids and plasmids mixed with microspheres, produced only limitedsuccess.

A second approach using augmented hydrodynamic delivery coupled withultrasonic pulses, capable of disrupting lipid DNA complexes, resultedin limited improvements in transgene delivery. Nevertheless, theinventors found that hydrodynamic manipulation of the kidney, via therenal vein, resulted in the robust endocytic uptake of fluorescentlytagged albumin and virtually eliminated surgery-related deaths. Based onthese observations the inventors coupled hydrodynamic delivery with theuse of baculovirus vectors. It was thought that the combination of abaculovirus vector in a relatively low titer would potentiallyfacilitate endocytic virus incorporation and minimize resultingtoxicity.

The GFP and Actin-GFP baculovirus vectors were then introduced intorodent kidneys using renal vein-guided, retrograde, pressurizedinjections. Transgene expression was then examined in these kidneys inlive animals with intravital multiphoton fluorescence microscopy, and intissue sections with confocal laser scanning microscopy. From these invivo studies, transgene expression was detected within 24 hours ofdelivery, and the kidneys appeared to recover from the mild ischemicevents (generated from the injection process) 3 days post transgenedelivery. At that time point the inventors observed robust and lengthyglomerular, tubular and vascular transgene expression, generated from asingle dose of low concentrations baculovirus injections.

Plasmid-derived transgene expression, generated from hydrodynamicinjections coupled with vascular clamping, generated efficient, stableand widespread transfection with intact renal structure and function.The vast improvement in superficial cellular transformation will readilyfacilitate live renal studies. Moreover, the ability to utilize plasmidDNA for animal models offers the benefit of having a potent vector witha great safety profile and level of biocompatibility. Plasmids can alsobe used to readily generate large volumes of a wide palate of exogenoustransgenes at relatively low costs to express.

These in vivo observations were confirmed by fixed tissue studies. Inthese studies robust signs of transgene expression were observed bothsuperficially and within deep medullary compartments. Again, bothnon-specific, and actin-targeted and tubulin-targeted GFP expression wasobserved in cortex and medulla. Diffuse cytosolic expression wasobserved throughout cells infected with the GFP encoding vectors wasobserved. Likewise, increased GFP-based fluorescence originating fromthe cytoskeletal and apical brush border segments in cells infected withthe actin-targeting vectors. The improved quality of thebaculovirus-based protein expression can support the use of thistechnique for in vitro studies.

Overall, this simplified method provides an ability to rapidly andreliably deliver multiple types of exogenous genes to various nephronsegments. Such a process increases widespread transgene expression.Without being bound by any particular theory, the observed transientincreases in pressure may be sufficient to facilitate transgene uptakeby basolateral anionic transporters and renal mechanotransduction, viathe delivery of transgenes through stretch-gated ion channels.Alternatively, the non-specific affinity of plasmid DNA in a stand-aloneform or bound to sera proteins, post its venous infusion, may benefitfrom enhanced endocytic uptake. This uptake may be triggered by rapidincreases in fluid volumes, throughout the kidney.

Hydrodynamic transgene delivery also has side effects, which result inbrief, mild, and reversible levels of tissue injury in live animals.This method allows one to modify renal segments at a measurable rate,while not inhibiting innate organ function. With the careful selectionof reporter constructs this method provides a medium to simultaneouslycontrast and examine innate and abnormal cells/structures. Moreover,this method builds on the tradition of techniques like micro-puncturetransgene delivery, as it enables similar live delivery and monitoring,while providing widespread expression of biochemically relevanttransgene concentrations.

Hydrodynamic-based cell transformation offers an attractive alternativeto transgenic models, and may be used as a research tool for the studyof normal and pathophysiological conditions in live mammalian systems.This method coupled with intravital multiphoton microscopy offers nearreal-time sub-cellular resolution. Thus, hydrodynamic cavitation hasclinical utility in a strategy for genetic therapy.

The present invention provides methods to rapidly deliver exogenousgenes, provide high-efficiency gene transfer and exceptional expressionlevels, along with monitoring methods related to their expression inlive mammalian kidneys. Previous methods described in the literaturehave produced inconsistent or very limited expression, have requiredspecialized equipment, were technically challenging to perform orrequired a tremendous commitment of time and resources in developing newanimal strains. The methods are relatively easy for any reasonablyskilled surgeon to perform, achieve consistent expression from procedureto procedure, provides relatively widespread and reasonably long-livedeffects in the kidney, and provides minimal injury to the kidney. Theinventors believe that the procedure described satisfies these criteriain that it provides for: 1) a viable infusion site and vascularmanipulations to affect widespread transgene delivery; 2) a significantdegree of vector uptake by several renal cell types; and 3) limitedgeneral injury and vector derived toxicity.

The innate structural barriers within the kidney pose significantobstacles to the delivery of exogenous genetic material to a variety ofrenal compartments. Delivery to the tubular epithelial cells, comprisinga significant fraction of the renal parenchyma and a key target in manystudies, has proved particularly challenging, due to the vascularmicroanatomy of the organ and the obstacle imposed by the glomerularfiltration barrier on access to the tubule lumen. These considerationsof tissue architecture probably account for the widely acknowledgedfailure of approaches such as systemic infusions of viral and plasmidvectors as useful methods for targeting most cells of interest in thekidney.

Straightforward surgical procedures allow easy access to the renalartery and vein and to the ureter and, in principle, any of the threevessels could provide a feasible access point for hydrodynamic delivery.However, the inventors found that injection into the renal artery provedunsuccessful due to the difficulty in achieving hemostasis withoutconcomitantly inducing an appreciable ischemic injury to the organ. Incontrast, using the renal vein as is described in the present inventionproved to be surprisingly successful in achieving widespread expressionof the fluorescent proteins used in the experiments.

The studies demonstrate that hydrodynamic forces produced by theinjection into the vein allow macromolecules to breach barriers thatnormally circumscribe their passage through the kidney. High molecularweight dextrans could be easily observed in the tubule lumen, as couldalbumin. An explanation for this observation is that the glomerularfiltration barrier is somehow breached by the hydrodynamic forces in theglomerulus that result from the injection. However, it is hard toconceive that these forces could be a simple increase in the pressure inthe glomerular capillaries producing a failure in the barrier, since itis unlikely that delivery at the renal vein could produce an increase inpressure at the glomerulus outside the normal tolerance of the system.It is possible that other routes of access to the tubular epithelialcells are possible. These include access to the basal side of the cellsvia the peritubular capillaries, or possibly a breach of the tightjunctions between the cells, which also provides an alternativemechanism to account for their observed appearance in the tubule lumen.

Whatever the mechanism, it is clearly transient, since only largemacromolecules present in the vasculature at the time of the injectionappeared to be able to access the tubule lumen or transfect the bulk ofthe cells in the kidney. It is reassuring for the potential utility ofthis technique that the physical effects of the injection are soshort-lived. The effect also appeared to be entirely confined to thekidney whose renal vein was injected, since the contralateral kidney andother highly vascular organs appeared to be completely unaffected. Therequirement for proximate delivery of the injection also accounts forthe failure of systemic delivery methods to achieve the same results,even those using hydrodynamic delivery.

The method was particularly successful in achieving transfection oftubular epithelial cells. All segments of the nephron showed expressionof the fluorescent proteins, with expression particularly prominent inthe proximal and distal convoluted tubules. Other cell types alsoexpressed the fluorescent proteins more sporadically, including cells inthe glomerulus and the tubular interstitium. Cell-type specificexpression of particular transgenes will require the use of specificpromoters, and it is possible that a ureteral delivery method may bemore optimal to efficiently target specific cell types.

The vectors used for delivery of the transgenes are a critical parameterin the success of efforts to express exogenous genes in the kidney. Thehigh efficiency of viral infection has made these vectors a favorite ofinvestigators in other fields, yet the inventors achieved essentiallyequal efficiency using plasmid vectors or adenovirus. Given the ease ofpreparation of plasmid vectors and the lesser degree of safety concernssurrounding their use compared to viral vectors, this is a very valuableaspect of this method.

Expression of the fluorescent proteins that were followed over a longertime course was remarkably persistent. There was only a moderate andprogressive decline in the level of expression over a four-week period.Since the inventors did not use vectors designed specifically forintegration into the host genome, incorporation of the sequences waspresumably sporadic and infrequent. However, in the healthy adult kidneythe rate of cellular turnover is thought to be relatively slow, and thismay account for the fairly long-lived expression observed in thestudies.

Baculovirus vectors produced the lowest efficiency of expression in thestudies. The inventors have not investigated the reason for thediscrepant behavior of these two systems, which may relate tocompatibility with host cell surface molecules necessary for virus entryin the rat system. The baculovirus vectors also seemed to compromise thestructure and function of cells that did become infected, as theinventors observed abnormal tubular morphology and fluorescent proteinaggregates in cells that did exhibit expression. This contrasted withthe observations with the plasmid and adenoviral vectors, where not onlywas tissue morphology normal in expressing regions but also the cellswere clearly viable and metabolically active, as judged by their abilityto actively internalize fluorescent dextrans from the tubule lumen.

It is desirable to provide methods in which long-term injury to thekidney is minimal. Such injury could severely compromise the outcome offuture studies. Ischemic injury to the kidney is a serious potentialcomplication, since the procedure involves a brief period of hemostasis.Ischemic injury could clearly be observed in experiments where bloodflow to the kidney was halted for more than 5 minutes, with theformation of debris or casts in the tubule lumen and sluggishmicrovascular flow in the peritubular capillaries. No such indicationsof injury were observed in the typical procedure, in which the vesselsare clamped for only ˜3 minutes or less. Good technique is thus clearlyimportant, but the inventors believe this should be easy for a practicedsurgeon to acquire. Investigators using this method should alsocarefully check for signs of injury using standard methods.

The inventors tried a number of more complex approaches. These includedcoupling hydrodynamic injections with ultrasonic pulsation, applied toenhance the disruption of lipid DNA complexes, or combining plasmid DNAwith microspheres. None of these complex approaches augmented proceduresenhanced the efficiency of expression compared to hydrodynamic deliveryalone.

Widespread, stable and lengthy transformation recorded in variousvascular, tubular and glomerular cell types accompanied intact renalstructure and function. This vast improvement in superficial cellulartransformation may be used to facilitate live renal studies that can bedirected towards understanding and treating the underlying causes ofrenal disease.

The similar levels of expression obtained from both non-viral and viralvectors, which were limited to the kidneys that received hydrodynamicinjection (no signs of expression were recorded in other organs posttransgene delivery), outline the versatility of the gene delivery methodfor kidney-targeted gene transfer. Moreover, hydrodynamic delivery mayalso facilitate long-term investigations using helper-dependent or 3rdgeneration adenovirus systems that do not express capsid proteins andprovide prolonged transgene expression.

However, in the case where the potential for mutagenesis derived over along-term may be an issue, as has been reported with recombinantadenovirus systems, the ability to utilize plasmid DNA for animal modelsand human gene therapy offers the benefit of having a potent vector witha great safety profile and level of biocompatibility. Plasmids can alsobe used to readily generate large volumes of a wide palate ofinexpensive exogenous transgenes.

Overall, this simplified method provides an ability to rapidly andreliably deliver multiple exogenous genes to various nephron segmentswith minimal injury. The uncharacteristic apical and basolateralincorporation, and filtration of large dextran molecules, as well asfluorescent protein expression observed in podocytes and epithelialcells of the S1 segment of proximal tubules may provide evidence thatsingle hydrodynamic injections can facilitate their transient passageacross the glomeruli filtration barrier.

Plasmid DNA (possible bound to sera proteins) and adenovirions maybenefit from enhanced endocytic uptake (primarily in the tubules),triggered by rapid increases in renal fluid volume after their venousinfusion. This technique provides large molecules the ability to accessthe lumens, and apical and basolateral borders of renal tubularepithelial cells.

It should also be noted that hydrodynamic transgene delivery also hasside effects, which result in brief, mild, and reversible levels oftissue injury in live animals. This method allows one to modify renalsegments at a measurable rate, while not inhibiting overall innate organfunction. With the careful selection of reporter constructs this methodcan provide a medium to investigate real time subcellular events invivo. Moreover, this method builds on the tradition of techniques likemicro-puncture transgene delivery, as it enables similar live deliveryand monitoring, while providing widespread expression of biochemicallyrelevant transgene concentrations.

In conclusion, hydrodynamic-based cell transformation offers anattractive alternative to transgenic models, and may also be used as aresearch tool for the study of normal and pathophysiological conditionsin live mammals. This method coupled with intravital two-photonmicroscopy offers near real-time sub-cellular resolution. Thus,hydrodynamic retrograde pressurized fluid delivery may have futureclinical utility as a strategy for human genetic therapy.

The present invention provides a simplified technique to rapidly induceand monitor transgene expression in live rat kidneys without significantinjury. To achieve this aim the inventors utilized two-photon excitationand confocal laser scanning microscopy techniques to investigatehydrodynamic venous delivery of vectors, including plasmids,baculovirions, and adenovirions.

Using pressurized renal vein injections of plasmid DNA the inventorsdeveloped a method to produce robust exogenous protein expression in arenal injury model. Transgene expression was recorded in live rats withmild and moderate ischemia/reperfusion renal injury that received thehydrodynamic treatment 1 and 24 hours after injury. These resultsprovide a novel platform to potentially facilitate the future study andmanagement of AKI during the initial phase of injury and at the time ofmaximal damage.

Hydrodynamic fluid delivery addresses the problem of reduced kidneyfunction in acute ischemia/reperfusion injury by providing substantialreductions in sera creatinine levels with a single retrograde infusioninto the left renal vein of rats with acute ischemia/reperfusion injury.These results provide an exciting platform to potentially facilitate thefuture study and management of AKI prior to a disease state, and at thetime of maximal injury (24 hours after the underlying insult occurs) inan attempt to limit or reverse such injuries.

Nucleic Acid Molecules

The nucleic acid molecule may encode, for example, a therapeutic proteinor an RNAi cassette, such as a shRNA. Alternatively, the nucleic acidmolecule may be used to repair or replace an endogenous gene, forexample DNA used for homologous recombination, or an oligonucleotideused for gene repair. Modifications include, for example, modifyingexpression levels of the gene and/or replacing a mutant gene with awild-type copy of the gene. The nucleic acid molecule may be DNA or RNA,including microRNA. Also preferably, the nucleic acid molecule is a DNAconstruct, in particular a cDNA or synthetic DNA, and can be furthermodified to improve transcription and/or translation in the host cell,or to reduce or minimize gene silencing. The nucleic acid moleculeconstruct may comprise, operably linked, a promoter region, anucleotide, and optionally, a termination signal. Preferably, thisconstruct is part of a plasmid. Preferably, the cells or tissue arestably transfected so that the transplanted cells or tissue may act, forexample, as a bio-factory to produce a therapeutic protein for a longperiod of time.

Multiple nucleic acid molecule sequences can be introduced into thecells or tissue, including multiple copies of the same nucleic acidmolecule sequence and/or multiple copies of differing nucleic acidmolecule sequences encoding for different therapeutic or markerproteins. In one embodiment, each nucleic acid molecule sequence ispresent on a separate polynucleotide construct, plasmid, or vector. Inanother embodiment, both nucleic acid molecule sequences are present onone polynucleotide construct, plasmid, or vector, with each sequenceunder the control of a separate promoter. Alternatively, and in yetanother embodiment, both nucleic acid molecule sequences are present onone polynucleotide construct, plasmid, or vector, with thepolynucleotide structured so that it is bicistronic and where bothnucleic acid molecule sequences are under the control of a singlepromoter. These various embodiments are further described below.

With respect to the embodiments where two differing nucleic acidmolecule sequences are present on one polynucleotide construct, plasmid,or vector, each sequence can be under the control of a separate promoteror can be under the control of a single promoter. In addition to a firstnucleic acid molecule sequence encoding for a selected therapeuticprotein, in this embodiment, a second nucleic acid molecule sequenceencoding, for example, a second therapeutic protein or a marker isincluded in the construct. Expression of this gene may be constitutive;in the case of a selectable marker this may be useful for selectingsuccessfully transfected cells or for selecting cells or transfectedpopulations of cells that are producing particularly high levels oroptimal therapeutic levels of the protein. It will also be appreciatedthat a selectable marker may be used to provide a means for enrichingfor transfected cells or positively selecting for those cells which havebeen transfected, before reintroducing the cells into the patient, aswill be described below.

Markers may include selectable drug resistance genes, metabolic enzymegenes, fluorescent proteins, bioluminescent proteins, or any othermarkers known in the art. Exemplary fluorescent proteins include, butare not limited to: green fluorescent protein, cyan fluorescent protein,yellow fluorescent protein, DsRed fluorescent protein, AsRed fluorescentprotein, HcRed fluorescent protein, and maxFP-green protein. When amarker gene is included in the vector construct, it will be appreciatedthat the marker can be used to quantify the amount of fluorescence aftertransfection and/or before transplantation and/or after transplantation.Quantitative determination of fluorescence can be undertaken aftertransfection but before transplanting the tissue using, for example,fluorescence microscopy, flow cytometry, or fluorescence-activated cellsorting (FACS) analysis, in order to quantify the expression offluorescence markers ex vivo. After transplanting the tissue, in vivomonitoring of the extent of fluorescence, as a measure of production ofthe therapeutic protein, can be done by examining the patient with afluorescent ophthalmoscope or a surgical microscope equipped forfluorescence imaging, and can be documented with a CCD camera. It willbe appreciated that the marker gene can be used to indicate levels oftransgene expression and can be monitored by a non-invasive or aminimally invasive procedure. If marker gene expression decreases,another tissue implant can be inserted into the patient to increase thelevel of therapeutic protein. By using a marker gene, diminishedexpression of the therapeutic protein can be recognized early, ratherthan waiting until decreased levels of the therapeutic gene lead todisease progression.

It will be evident that for many gene therapy applications, selectionfor expression of a marker gene may not be possible or necessary. Also,it is possible that for in vivo applications, vectors without anyinternal promoters may be preferable. Single transcription unit vectors,which may be bicistronic or poly-cistronic, coding for one or two ormore therapeutic genes, may be designed.

Where two or more genes are present and under transcriptional control ofa single promoter, there may be an internal ribosome entry site (IRES),e.g. from picornaviral RNA, to allow both genes to be separatelytranslated from a single transcript. Retroviruses incorporating IRESsequences are known in the art, for example in U.S. Pat. No. 5,665,567.Briefly, in bicistronic or multicistronic vectors, the individualreading frames of the gene segments encoding the proteins lie on thetranscription unit (expression unit). Expression of each cistron iseffected using a single promoter, in conjunction with a specific nucleicacid molecule sequence, typically untranslated regions of individualpicorna viruses, e.g. poliovirus or encephalomyocarditis virus, or acellular protein, e.g. BiP. In the picorna viruses, a short segment ofthe 5′ untranslated region, the so-called IRES (internal ribosomal entrysite) functions as an initiator for translation of reading frames.

By way of a specific example, the cells or tissue can be transfectedwith a plasmid having one promoter that drives the expression of a firsttherapeutic protein, such as pigment epithelium-derived factor (PEDF),and of a selectable marker, such as a fluorescent protein like enhancedgreen fluorescent protein (eGFP) under control of a cytomegalovirus(CMV) promoter. The CMV promoter is positioned at the 5′ end of theconstruct. Downstream of the 3′ end of the CMV promoter is the PEDFnucleotide sequence that encodes for PEDF protein. In the 3′ directionof PEDF is an IRES site, which is designed to allow translation ofmultiple genes on an mRNA transcript. Following the IRES site in the 3′direction is the eGFP coding sequence. The IRES will allow translationof eGFP as well as translation of PEDF.

The promoter region of the construct can be chosen from among allpromoter regions that are functional in mammalian cells, in particularhuman cells. The promoter can be a strong or weak promoter, aconstitutive or a regulated/inducible promoter, a ubiquitous orselective promoter. The promoter can be of different origin such ascellular, viral, artificial, and the like. Particular types of promotersare house-keeping promoters, i.e., promoters from cellular genesexpressed in mammalian tissues or cells, or viral promoters (CMV, LTR,SV40, etc.). Furthermore, the promoter region can be modifiedartificially to include enhancer element(s), inducibility element(s) andthe like. The promoter, secretion and termination region sequences canbe selected and adapted by the skilled artisan based on the polypeptide,the pathology, the vector used, etc. In this regard, the nucleic acidmolecule construct can be inserted into various kinds of vectors such asplasmids, episomes, artificial chromosomes and the like.

The nucleic acid molecule construct can optionally include a secretionsignal, positioned between the promoter and coding regions, whichallows, or facilitates, the secretion of the polypeptide outside of thecells. The secretion signal may be homologous with respect to thepolypeptide (i.e., from the same gene) or heterologous thereto (i.e.,from any other gene encoding a secreted polypeptide, in particular amammalian gene, or artificial). Examples of secretion signals includethe signal peptide of vascular endothelial growth factor (VEGF), pre pronerve growth sequence (NGS), and the like.

Various approaches may be used to achieve long-term expression of thenucleic acid molecule in the cells or tissue. One approach involves acircular vector carrying a recombination site and the polynucleotidesequence encoding for the therapeutic protein, shRNA, miRNA, etc., andthe transfection is accompanied by introduction of a recombinase thatfacilitates recombination between the vector's recombination site and asecond recombination site in the genome of the cell being transfected.Constructs carrying a recombination site, such as a phiC31 attB site,have been described. It will be appreciated, however, that other meansfor long-term gene expression are contemplated, such as the othermembers of the serine recombinase family, transposases (e.g., “SleepingBeauty”), DNA mini-circles, plasmids optimized for minimal genesilencing, or the use of a stable extrachromasomal vector such as EBV.When using a phiC31 attB recombination site, the nucleic acid moleculeconstructs are comprised of the phiC31 integrase system to achievesite-specific integration into a target genome of interest.

Bacteriophage phi-C31 integrase recognizes pseudo-recombination sitespresent in eukaryotic cells. For genetic manipulation of a eukaryoticcell, phiC31 integrase and a vector carrying a phiC31 wild-typerecombination site are placed into the cell. The wild-type recombinationsequence aligns itself with a sequence in the eukaryotic cell genome andthe phiC31 integrase facilitates a recombination that results inintegration of a heterologous gene into the eukaryotic genome. It iscontemplated that any attB site, any attP site, or any pseudo att siteis present on any nucleotide sequence used to introduce genetic materialinto the genome of the harvested or cultured cells.

Accordingly, in one embodiment, the method of integrating apolynucleotide sequence into a genome of a cell comprises introducinginto the cell (i) a circular targeting construct, comprising a firstrecombination site and a polynucleotide sequence of interest, and (ii) aphiC31 integrase, native or modified, wherein the genome of the cellcomprises a second recombination site (ie. a pseudo att site) native tothe human genome. Recombination between the first and secondrecombination sites is facilitated by the site-specific integrase.

The therapeutic gene and the attB sequence are preferably introducedinto the target cell as circular plasmid DNA. The integrase may beintroduced into the target cell (i) as DNA encoding the integrase on asecond plasmid, (ii) mRNA encoding the integrase, or (iii) inpolypeptide form. Once phiC31 is introduced into the cell, the cell ismaintained under conditions that allow recombination between the firstand second recombination sites and the recombination is mediated by thephiC31 integrase. The result of the recombination is site-specificintegration of the polynucleotide sequence of interest in the genome ofthe cell.

Transfection of a wide variety of genes encoding for therapeuticproteins is contemplated, and preferred candidate genes include genesthat encode for diffusible proteins that act extracellularly to have atherapeutic effect.

In some embodiments, the vector is a viral vector. “Viral vector” refersto recombinant viruses engineered to effect the introduction ofexogenous nucleic acid molecules into cells. Viral vectors include, forexample, retroviruses, adenoviruses, adeno-associated viruses (AAV),baculoviruses, vaccinia viruses, herpes viruses, alphavirsus vectors,alphavirus replicons and lentivirus vectors.

In specific embodiments, the viral vector may be a baculovirus vector.Baculovirus vectors, such as, for example, those derived from AutographaCalifornica Multicapsid Nucleopolyhedrovirus (AcMNPV) are useful in thepresent invention.

A person skilled in the art would readily appreciate how to constructbaculoviral vectors for use in the invention. Recombinant baculovirusvectors may be constructed according to instructions accompanyingcommercial baculovirus expression systems, for example, the Bac-to-Bac™Expression system (Invitrogen). Recombinant baculoviral vectors may bemodified by molecular biological techniques, including PCR-basedtechniques and other cloning techniques, as will be known to a skilledperson and described, for example, in Sambrook et al., Molecular CloningA Laboratory Manual (3rd ed.), Cold Spring Harbour Press.

Viral vectors may be engineered to contain increased levels of the viralenvelope glycoprotein gp64. Recombinant viral vectors may also bemodified by incorporating foreign envelope proteins into the envelope ofthe viral virion. For example, increased neural infection efficiency maybe achieved by pseudotyping rabies virus glycoprotein (RVG) or vesicularstomatitis virus G protein (VSVG), herpes envelope glycoprotein orenvelope proteins derived from .alpha.- or rhabdovirus into the envelopeof the viral virion. Alternatively, the cell specificity of viralinfection may be increased by incorporating antibodies directed againstcell-specific protein receptors into the viral envelope.

To minimize or avoid any possibility for inactivation by serumcomplement, recombinant viruses may be modified to increase theirresistance to the complement system, including, for example, byincorporating human decay-accelerating factor into a viral envelope.

In other embodiments, the vector is a non-viral vector. “Non-viralvectors” refers to systems other than viral vectors that may be used tointroduce exogenous nucleic acid molecules, for example plasmids, into acell. Non-viral vectors include, but are not limited to polymer-based,peptide-based and lipid-based vectors. Many non-viral vectors arecommercially available, such as, for instance PEI 25K (Sigma-Aldrich,St. Louis, Mo.) Lipofectamine™ 2000 (Invitrogen, Carlsbad Calif.).Complexes of these vectors and nucleic acid molecules may be preparedaccording to commercial instructions, or by following protocols known toa person skilled in the art, such as, for example, Boussif et al. (1995,Proc. Nat. Acad. Sci. 92:7297).

Generally, non-viral gene-delivery systems rely on the direct deliveryof the target nucleic acid molecule or on nonspecific internalizationmethods. Non-viral gene delivery systems and methods for theirtransfection would be known to a person skilled in the art, and include,for example, naked plasmids, DEAE-dextran, calcium phosphateco-precipitation, microinjection, liposome-mediated transfection,cationic lipids, and polycationic polymers. As would further beappreciated by a person skilled in the art, some of these methods, suchas, for example, microinjection, liposome-mediated transfection,polycationic polymers, are capable of transfecting cells both in vivoand in vitro. These non-viral vectors may be modified to enhancenerve-specific transfection, for example by linking the vector to one ormore ligands that may specifically or preferentially bind to neuronalcells. For example, nerve-specific transfection of polylysine/DNAcomplexes may be obtained by covalently linking the nontoxic fragment Cof tetanus toxin to polylysine.

Non-viral vectors containing DNA with bacterial sequences often haveincreased palindromic CpG sequences relative to eukaryotes, and theseforeign CpG sequences may serve as strong immunostimulatory agents invertebrates. Reducing CpG content therefore may be advantageous and mayalso enhance protein expression as CpG sequences may be methylated ineukaryotic hosts, which can result in the transcriptional silencing. Insome embodiments, the CpG content of the DNA of non-viral DNA-basedvectors is reduced. A person skilled in the art would readily appreciatethat the CpG dinucleotide content of a vector may be reduced usingstandard molecular biology techniques, such as oligonucleotide orPCR-based mutagenesis as described, for example, in Chevalier-Marietteet al. 2003, Genome Biology 4:R53.

The transcriptional activity of a promoter in some instances may beweak, providing a less than ideal level of expression of therapeuticgene sequences. In various embodiments, the promoter may be operablylinked to an enhancer. As would be understood by a skilled person, an“enhancer” is any nucleotide sequence capable of increasing thetranscriptional activity of an operably linked promoter and, in the caseof a neuron-specific promoter, of selectively increasing thetranscriptional activity of the promoter in neuronal cells. A number ofenhancers are known and a person skilled in the art would also know howto screen for novel enhancer sequences, for instance, by screeningnucleotide sequences capable of increasing the transcription of areporter gene, for instance, through functional mapping.

A first nucleic acid molecule sequence is operably linked with a secondnucleic acid molecule sequence when the sequences are placed in afunctional relationship. For example, a coding sequence is operablylinked to a promoter if the promoter activates the transcription of thecoding sequence. Similarly, a promoter and an enhancer are operablylinked when the enhancer increases the transcription of operably linkedsequences. Enhancers may function when separated from promoters and assuch, an enhancer may be operably linked to a promoter even though it isnot contiguous to the promoter. Generally, however, operably linkedsequences are contiguous.

In different embodiments, the enhancer may be a heterologous enhancer,meaning a nucleotide sequence which is not naturally operably linked toa promoter and which, when so operably linked, increases thetranscriptional activity of the promoter. Reference to increasing thetranscriptional activity is meant to refer to any detectable increase inthe level of transcription of an operably linked sequence compared tothe level of the transcription observed with a promoter alone, as may bedetected in standard transcriptional assays, including those using areporter gene construct.

The enhancer may be a known strong viral enhancer element such as Roussarcoma virus (RSV) promoter, SV40 promoter, CMV enhancer or promoterincluding CMV immediate early (IE) gene enhancer (CMVIE enhancer).

In different embodiments, the vector comprises a gene encoding a markerprotein whose expression and cellular or subcellular localization maybereadily determined. “Marker protein” refers to a protein whose presenceor subcellular localization may be readily determined, such as a greenfluorescent protein (GFP) or any of its enhanced derivatives. Othermarker proteins would be known to a person skilled in the art. Indifferent embodiments, the gene may encode an enzyme whose expressionmay be readily determined by providing a specific substrate anddetecting the products of enzymatic turnover, such as, for example, byproviding luciferin to cell or cell lysates containing luciferase. Inother embodiments, the marker protein may be any protein whoseexpression may be detected immunologically, for example by providing alabeled antibody that specifically recognizes the marker protein. Theantibody is preferably a monoclonal antibody and may be directly orindirectly labeled according to methods known in the art, such as, forexample, labeling with a fluorescent dye and detecting expression of theprotein by fluorescence microscopy. Other immunological detectionmethods, including without limitation, immunogold staining,radiolabelling, colorimetric enzymatic precipitation would be known to aperson skilled in the art.

Preferably, the vector comprises a therapeutic gene or a therapeutictransgene whose expression produces a therapeutic product. The term“gene” is used in accordance with its usual definition, to mean anoperatively linked group of nucleic acid sequences. As used herein,“therapeutic product” describes any product that affects a desiredresult, for example, treatment, prevention or amelioration of a disease.The therapeutic product may be a therapeutic protein, a therapeuticpeptide or a therapeutic RNA, such as, for example, a small interferingRNA (siRNA), microRNA or an anti-sense RNA.

To aid in administration, the vectors may be formulated as an ingredientin a pharmaceutical composition. The compositions may routinely containpharmaceutically acceptable concentrations of salt, buffering agents,preservatives and various compatible carriers or diluents. For all formsof delivery, the vectors may be formulated in a physiological saltsolution.

The proportion and identity of the pharmaceutically acceptable diluentis determined by chosen route of administration, compatibility with thevector and standard pharmaceutical practice. Generally, thepharmaceutical composition will be formulated with components that willnot significantly impair the biological activities of the vector.Suitable vehicles and diluents are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985).

Solutions of the vectors may be prepared in a physiologically suitablebuffer. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms, but thatwill not inactivate the vector. A person skilled in the art would knowhow to prepare suitable formulations. Conventional procedures andingredients for the selection and preparation of suitable formulationsare described, for example, in Remington's Pharmaceutical Sciences andin The United States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999.

In some embodiments, the vectors are administered to a vertebrate host.In a specific embodiment, the vectors are administered to a human host.

Effective amounts of vectors can be given repeatedly, depending upon theeffect of the initial treatment regimen. Administrations are typicallygiven periodically, while monitoring any response. It will be recognizedby a skilled person that lower or higher dosages may be given, accordingto the administration schedules and routes selected.

When administered to a human patient, for example, the vectors areadministered in an effective amount and for a sufficient time period toachieve a desired result. For example, the vectors may be administeredin quantities and dosages necessary to deliver a therapeutic gene, theproduct of which functions to alleviate, improve, mitigate, ameliorate,stabilize, prevent the spread of, slow or delay the progression of orcure a peripheral neuronal neuropathy.

The effective amount to be administered to a patient can vary dependingon many factors such as, among other things, the pharmacodynamicproperties of the therapeutic gene product, the mode of administration,the age, health and weight of the subject, the nature and extent of thedisorder or disease state, the frequency of the treatment and the typeof concurrent treatment, if any. In embodiments employing viral vectors,the effective amount may also depend on the virulence and titer of thevirus.

One of skill in the art can determine the appropriate amount based onthe above factors. Vectors may be administered initially in a suitableamount that may be adjusted as required, depending on the clinicalresponse of the patient. The effective amount of a vector can bedetermined empirically and depends on the maximal amount of the vectorthat can be safely administered. In some embodiments, the vector mayhave little cytotoxicity in vertebrates and may be administered in largeamounts. However, the amount of vectors administered should be theminimal amount that produces the desired result.

In various embodiments, a dose of about 10⁹ recombinant baculovirusparticles are administered to a human patient. In other embodiments,about 10² to about 10⁹ recombinant baculovirus particles, about 10⁶ toabout 10⁹ recombinant baculovirus particles, about 10² to about 10⁷recombinant baculovirus particles, about 10³ to about 10⁶ recombinantbaculovirus particles, or about 10⁴ to about 10⁵ recombinant baculovirusparticles may be administered in a single dose. In some embodiments, thevector may be administered more than once, for example, by repeatedinjections. In other embodiments, the viral vector may be repeatedlyadministered.

While a number of exemplary aspects and embodiments are discussedherein, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations therefore. It is thereforeintended that the following appended claims hereinafter introduced areinterpreted to include all such modifications, permutations, additionsand sub-combinations are within their true spirit and scope. Eachapparatus embodiment described herein has numerous equivalents.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.Whenever a range is given in the specification, all intermediate rangesand sub-ranges, as well as all individual values included in the rangesgiven are intended to be included in the disclosure. When a Markushgroup or other grouping is used herein, all individual members of thegroup and all combinations and sub-combinations possible of the groupare intended to be individually included in the disclosure.

EXAMPLES Example 21 Hydrodynamic Methods for Transgene Expression inKidney Tissues

A. Materials and Methods

Cell Culture

Mouse Kidney Cell Culture. The inventors used epithelial cells from theS3 segment of the proximal tubules. These cells were cultured in mediumprepared by combining 500 ml of essential medium (Fisher Scientific,Pittsburgh, Pa.) with 7.5% of sodium bicarbonate, 7% of fetal bovineserum (FBS), and 1% of Pen-Strep, (Fisher Scientific, Pittsburgh, Pa.).The cells were grown in a 37° C., 5% CO₂, 38% O₂ humid incubator.

MDCK Cell Culture. Madin-Darby Canine Kidney (MDCK) strain II cells,were grown in minimal essential media (Fisher Scientific, Pittsburgh,Pa.) with 8% fetal bovine serum, 1% L-glutamine, penicillin/streptomycin(Fisher Scientific, Pittsburgh, Pa.) and hygromycin (Calbiochem, SanDiego, Calif.), and kept in a 37° C., 5% CO₂ humid incubator.

Rats

Male and female Sprague Dawley (Harlan Laboratories, Indianapolis, Ind.)and Munich Wistar rats (Frömter and Simonsen strains of Wistar rats werea gift of Dr. Bruce Molitoris, Indiana University School of Medicine),ranging in weight from 150 to 470 gm, were used for these studies. Therats were given free access to standard rat chow and water throughoutthe studies. All experiments were conducted in accordance with theNational Institutes of Health Guidelines and were approved by theIndiana University School of Medicine Institutional Animal Care and UseCommittee (IACUC).

Dyes and Fluorescent Probes

Tolonium Chloride. The inventors prepared stock solutions by dissolving50 mg of tolonium chloride dye (Toluidine Blue O, Electron MicroscopySciences, Fort Washington, Pa.), in 5 ml of 0.9% saline. 0.5 ml of thismixture was used for each hydrodynamic injection.

Albumin, Dextrans and Hoechst. The following fluorescent probes wereused in the intravital two-photon fluorescent imaging studies: Texas Redlabeled albumin in phosphate buffered saline (PBS) prepared by combingTexas red sunfonyl chloride from (Life Technologies, Carlsbad, Calif.)and albumin fraction V powder (Sigma-Aldrich, St. Louis, Mo.), 3 kDaCascade Blue, 4 and 150 kDa Fluorescein Isothiocyanate (FITC) dextrans(Invitrogen, Carlsbad, Calif.); 150 kDa Tetramethyl RhodamineIsothiocyanate (TRITC) dextran (TdB Consultancy, Uppsala, Sweden); andHoechst 33342 (Invitrogen, Carlsbad, Calif.). The final albumin anddextran injection solutions were prepared from diluting 50 μl of each 20mg/ml stock solution in 0.5-1 ml of saline, and 30-50 μl of Hoechst wasdiluted in 0.5 ml of saline.

Transgene Vectors

Plasmid Vectors. Plasmid DNA was isolated using Qiagen Maxi Prep systems(Qiagen, Chatsworth, Calif., USA). These plasmids encoded: enhancedgreen fluorescent protein (EGFP), EGFP-actin and EGFP-tubulin (ClontechLaboratories, Inc., Mountain View, Calif., USA); EGFP-occludin (a giftfrom Dr. Clark Wells, Indiana University School of Medicine);H2B-tdTomato (a gift from Dr. Richard Day, Indiana University School ofMedicine). For hydrodynamic injections, the range of doses the inventorsused was 1-3 μg of plasmid DNA per gram of body weight diluted in 0.5 mlof saline.

Baculovirus Vectors. Cellular Light™ GFP, EGFP-actin and Null (control)BacMam 2.0 baculovirus expression vectors were from Life Technologies(Carlsbad, Calif.). The EGFP-actin baculovirus vector encodedfluorescent proteins with a human sequence targeting them to bothfilamentous and globular actin. The Null reagent lacks mammalian geneticconstituents, and is designed to identify potential baculovirus-mediatedeffects and distinguish fluorescence signals from innate tissuefluorescence. A range of doses was used, spanning 1×10⁵ to 1×10⁷ viralparticles/ml, suspended in saline.

Adenovirus Vectors. Replication-incompetent EGFP-actin and RFP-actinadenovirus vectors (gift of Dr. James Bamburg, Colorado StateUniversity), were kept at concentrations of 3×108 pfu/ml in DMEM at −80°C. For injections, the inventors used 3×10⁵ to 3×10⁷ pfu of eachadenovirus vector suspended in 0.5 ml of saline solution.

Retrograde Venous Hydrodynamic Injection

Rats were anesthetized by inhaled isoflurane (Webster Veterinary Supply,Inc., Devens, Mass.; 5% in oxygen), and then placed on a heating pad tomaintain core body temperature of 37° C. Temperature was monitored usinga rectal probe. The abdomen was shaved, cleaned with Betadine SurgicalScrub (Purdue Products L.P., Stanford, Conn.) and a midline incision wasmade to expose and isolate the left renal vein. The renal artery andvein were occluded with micro-serrefine clamps (Fine Science Tools(USA), Inc., Foster City, Calif.).

The vein was then elevated with either 3-0 or 4-0 silk suture thread(Fine Science Tools (USA), Inc., Foster City, Calif.). At that time 0.5ml of fluorescent probe or transgene expression vector solution wasinfused retrograde into the vein (i.e. towards the kidney) over a periodof approximately 5 seconds, using a 30-gauge stainless steel needleattached to a 1 ml syringe, at the site between the clamp and the kidney(FIG. 1A). The needle was removed, and pressure was applied to theinjection site using a cotton swab, to induce hemostasis. The vascularclamps were removed (the venous clamp was removed before the arterialclamp) to restore renal blood flow. The total clamping period lasted notmore than 3 minutes. After this, the midline incision was closed and theanimal was allowed to fully recover.

Monitoring Vital Signs During Renal Vein Hydrodynamic RetrogradeInfusions in Live Rats

The inventors made incision in the legs of anesthetized rats to exposefemoral arteries. The arteries were isolated with two 3-0 or 4-0 silkloops. Using mirco-serrefine clamps the inventors clamped off the arteryand tied off the loops as well. Each loop was then clamped with a pairof hemostats to stiffen and elevate each artery. The inventors then madea small incision in the femoral artery and inserted a PE-50 tubingcatheter into its lumen. The other silk loop was used to anchor thecatheter in place. This tubing was attached to a three-way port that waslinked to a PowerLab 8/30 data acquisition system (ADInstrumentsColorado Springs, Colo.) to record temperature, blood pressure and heartrate.

Fluorescence Microscopy

Intravital and Ex Vivo Two-photon Fluorescence Microscopy. Each rat wasgiven an intraperitoneal dose of 50 mg/kg pentobarbital and then placedon a heating pad to maintain a core body temperature of 37° C. Once theanimal was fully sedated, its left side was shaved and a vertical flankincision was made to externalize the left kidney. The kidney was thenpositioned inside a glass bottom dish containing saline, which was setabove either a 20× or 60× water immersion objective for imaging.Similarly, for ex vivo imaging, sagittal plane sections of kidneysharvested from anesthetized rats were positioned inside the glass bottomdish containing saline.

Fluorescent images were acquired using an Olympus (City, State) FV1000-MPE Microscope equipped with a Spectra Physics (City, State) MaiTaiDeep See laser, with dispersion compensation for two-photon microscopy,tuned to 770-860 nm excitation wavelengths. The system was also equippedwith two external detectors for two-photon imaging, and dichroic mirrorsavailable for collecting blue, green and red emissions. The system wasmounted on an Olympus IX81 inverted microscope. Bars in all figures are60 μm.

Jugular Vein Infusions

Each rat was first anesthetized by inhaled isoflurane (WebsterVeterinary Supply, Inc., Devens, Mass.), 5% in oxygen, and then given anintraperitoneal injection of approximately 50 mg/kg of pentobarbital.The rat was placed on a heating pad to maintain its core bodytemperature of 37° C. Once the animal was fully sedated, its neck wasshaved and it was restrained on a heating pad. An incision was made toexpose the jugular vein. The vein was isolated with two 3-0 or 4-0 silkloops. The loop closer to the animal's head was tied and clamped with apair of hemostats to stiffen and elevate this vein. A small incision wasthen made in the jugular vein to insert a PE-50 tubing catheter into itslumen. The other silk loop was used to anchor the catheter in place.This tubing was attached to a 1 ml syringe containing the solution thatwould be infused into the vein.

Confocal Laser Scanning Fluorescence Microscopy

Whole kidneys were harvested from live animals directly beforeeuthanasia. These kidneys were immersion fixed with 4% paraformaldehydesolution. After this, 100-200 μm thick sections were obtained using avibratome. These sections were then mounted onto glass slides and imagedwith the previously described Olympus IX81 inverted microscope inconfocal mode.

Estimation of Transgene Delivery Efficiencies

The inventors used two-photon microscopy to analyze the time course andspatial distribution of renal transgene expression. The inventorsestimated the transgene delivery efficiency for each vector in vivousing intravital fluorescent two-photon microscopy, and in vitro withconfocal laser scanning microscopy. Using two-photon microscopy theinventors determined the efficiency of transgene expression within livesuperficial cortex segments of several rats across a 28-day period aftertransgene delivery. The inventors began the measurements 3 days aftertransgene delivery, having previously determined that this was the pointwhen the inventors reproducibly observed signs of stable transformationand normal renal function.

For these efficiency measurements, the inventors set a threshold signalthat was above the highest observed autofluorescence level anddistinguished transgene expression from autofluorescent background. Theinventors determined that transgene fluorescence signals had intensitiesat least double those of autofluorescence signals. Using thesethresholds, the inventors then calculated the percentage of nephroncross-sections that expressed the reporter transgenes within fieldsacquired with the 60× objective. This final percentage (efficiencyvalue) was calculated as the average percentage of transfected(transduced) nephron cross-sections within 10 randomly chosen adjacentfields.

Similarly, the in vitro estimations allowed the inventors to determinethe degree of transgene distribution throughout all regions of thecortex and medulla, including those that are presently inaccessible byintravital two-photon microscopy. For these estimations the inventorsfirst collected a montage of fields using confocal laser scanningmicroscopy covering a wedge of the kidney from the renal cortex to thelevel of the pedicle. Thereafter, the inventors estimated the extent oftransformation using the same approach, within 100 μm×1000 μm regions.

Serum Creatinine Measurements

Creatinine levels were measured in serum samples obtained from rats usedin these studies, using the creatinine kinase reagent set (PointScientific, Inc., Canton, Mich.) in a Beckman Creatinine Analyzer 2(Beckman Instruments, Brea, Calif.) Values are reported in mg/dl45.

Measurement of Hydrodynamic Injection Parameters

To characterize the hydrodynamic delivery process, the inventorsmonitored time-dependent pressure profiles during the injection with adamped ultrasonic Doppler flowmeter (Model T206, Transonic Systems,Ithaca, N.Y.). A PE-50 polyethylene catheter tubing (Clay Adams,Division of Becton, Dickson and Company, Parsippany, N.J.), wasintroduced into the femoral vein and traversed to the level of thebifurcation adjoining the renal vein and inferior vena cava.

B. Widespread Fluorescent Protein Expression Observed in Various RenalSegments In Vivo, Ex Vivo and In Vitro

The inventors detected widespread and reproducible expression of avariety of fluorescent protein constructs delivered using thehydrodynamic method. The inventors observed a typical autofluorescentsignature and normal morphology in kidneys that were not injected orinjected with saline alone (FIGS. 2-8). Following hydrodynamic deliveryof plasmid/adenovirus vectors, the inventors observed abundantexpression of fluorescent proteins by in live kidneys (FIGS. 2-8). Thefluorescent protein signals (FIGS. 2-8) were at least double theintensity of the autofluorescence (FIGS. 2-8) and showed characteristicspectral distributions that clearly distinguished them from theendogenous autofluorescence. Widespread transgene expression wasobserved as early as 24 hours after hydrodynamic delivery. During thefirst 36 hours after transgene delivery the inventors did occasionallyobserve cellular debris within tubule lumens. Such tissue damage mayhave resulted from the hydrodynamic forces produced by the injection orfrom mild ischemia-reperfusion injury associated with the injectionprocess. However, this minimal injury completely subsided after thisperiod, and at 3 days after the injection the kidneys appeared to bestable without signs of injury. The inventors carried out furtherstudies to confirm that the kidney had not sustained significant injury(see below).

Expression of a variety of fluorescent proteins was observed within liveproximal and distal tubules (FIGS. 2-8); glomeruli (FIGS. 6B and 6C);the supporting interstitium (FIG. 6D); in adipose tissues at the surfaceof the kidney (FIG. 6E); and the renal capsule (FIG. 6F). Fluorescentprotein expression was not limited to the superficial cortex, but it wasnecessary to use confocal microscopy of fixed tissues from injectedanimals to document expression in these deeper regions, which arepresently inaccessible to two-photon intravital imaging. High levels ofexpression were found to extend across the cortex and medulla to thelevel of the papilla (FIG. 7B). Furthermore, it should be noted thatsingle hydrodynamic injections of a mixture of EGFP-actin and RFP-actinadenovirus vectors generated the simultaneous expression of bothfluorescent proteins, sometimes in the same cell, indicating that thismethod can be used for simultaneous expression of multiple genes.

The morphology of nephron segments expressing fluorescent proteins fromplasmid vectors appeared normal. Likewise, injections of adenovirusvectors (3×10⁵ pfu) resulted in stable transgene expression with normaltissue morphology. However, injections of higher titers of adenovirus(3×10⁶-3×10⁷ pfu) resulted in fluorescent debris/casts (within tubularlumens) that persisted beyond 3 days after viral delivery, indicating apossible immunological response to higher viral titers. In comparison,following the delivery of baculovirus vectors, areas that expressedfluorescent proteins generally deviated from normal tissue morphologyand showed fluorescent protein aggregation.

Images obtained from rats that received hydrodynamic injections ofplasmids that expressed EGFP-occludin and H2B-tdTomato fluorescentproteins provided clear signs of proper probe localization andmorphology. For instance, EGFP-occludin signals ran between adjacentnuclei as punctate fluorescent bands along regions that would correspondto tight junctions (FIG. 2J). Fluorescent histone protein signals fromH2B-tdTomato protein expression co-localized with nuclei counterstainedwith Hoechst (FIG. 2L).

Similarly, in images taken from rats injected with plasmids (FIG. 3), oradenovirus vectors containing EGFP-actin (FIGS. 4 and 5) and RFP-actin(FIG. 5), there was characteristic labeling of the brush border inproximal tubules that expressed these transgenes.

Transgene expression in the glomerulus was investigated primarily inWistar rats (FIGS. 6B and 6C). These rats have superficial glomerulithat are routinely accessible for imaging by two-photon microscopy. Theinventors also visualized glomerular transgene expression in a SpragueDawley rat on the rare occasion that this structure appeared within therange of two-photon imaging in this rat strain. Glomerular morphologywas grossly normal in rats that received hydrodynamic saline injections(FIG. 6A).

The appearance of fluorescent protein distribution was consistent withexpression in podocytes (FIG. 5B). Similarly, fluorescent proteinexpression was visualized in S1 segments of proximal tubules andparietal epithelial cells of the Bowman's capsule (FIG. 5C).Additionally, 150 kDa TRITC dextran molecules, introduced into thejugular vein of animals that had previously been subject to hydrodynamicplasmid delivery, were characteristically confined to the vasculature(FIGS. 5B and 5C). This provided further evidence of maintainedglomerular structural and functional integrity following transgenedelivery and expression.

Plasmid- and adenovirus-derived fluorescent protein expression was alsopresent in cells within the peritubular interstitium that had morphologysimilar to either endothelial cells or monocytes (FIG. 5D), as well asin cells adjacent to the renal capsule (FIG. 5F). Strikingly, no signsof fluorescent protein expression were found in the contralateral kidney(i.e. non-injected kidney) or the other highly vascular organs examined(heart, liver, lung and spleen).

Hydrodynamic Injections can Generate Efficient Levels of TransgeneExpression in Mammalian Kidneys

The inventors examined tissue sections harvested from rats 3 days afterthey were treated with plasmids, baculovirus and adenovirus vectors, togain insight into the efficiency of the hydrodynamic delivery method foreach type of vector. For this work the inventors used confocal laserscanning microscopy to visualize fluorescent protein expression inkidney sections encompassing the entire depth of the kidney, from thecortical surface to the level of the renal pedicle (FIG. 7B). Withplasmid or adenovirus vectors the inventors typically saw that multiplecells (greater than 50%) in a particular tubular cross-sectionsimultaneously expressed the fluorescent proteins. However, usingbaculovirus vectors the inventors frequently observed only single cellsexpressing the fluorescent proteins.

Baculovirus-based transformation provided the lowest deliveryefficiencies ranging from 10 to 50% of nephron cross-sections (FIG. 7C).In particular, within the most superficial cortical regions, which wouldbe accessible by intravital two-photon microscopy, there was a 10%efficiency. However, at depths greater than 500 μm there was a gradualdecrease in fluorescent protein expression in regions that wouldcorrespond to the deeper cortex, cortico-medullary junction and medulla.

Much higher levels of fluorescent protein expression were obtained usingplasmid and adenovirus vectors (FIG. 7C). Using these vectors, 40 to 86%of nephron segments showed fluorescent protein expression. Within thesuperficial cortex (less than 100 μm from the surface), the inventorssaw approximately 78-86% of nephron cross-sections expressingfluorescent proteins, explaining the relative ease with which expressionwas detected in live animals.

The high level of fluorescent protein expression in this superficialregion of the cortex permitted the inventors to investigate the level ofexpression as a function of time by imaging live animals over a 4-weekperiod. Over this period, the percentages of nephron cross-sectionsexpressing fluorescent proteins ranged from 80 to 14% using adenovirusvectors, and 61 to 28% with plasmid vectors (FIG. 7D). Thus, expressionappears to be relatively long-lived with even the rudimentary vectorsused in this study.

C. Nephron Structure and Function Appear Normal after HydrodynamicDelivery

The inventors looked for evidence of injury following hydrodynamic genedelivery by examining kidney structure and function using severalapproaches. In animals injected with high molecular weight dextrans (150kDa TRITC) via the jugular vein, the inventors observed robust perfusionof the peritubular vasculature and confinement of the dextran by theglomerular filtration barrier. The inventors extended this analysis bysimultaneously injecting high (150 kDa) and low (3 kDa) dextrans labeledwith TRITC and Cascade blue respectively via the jugular vein. Thisanalysis was conducted on rats from 3 to 28 days after they receivedhydrodynamic transgene injections of plasmids and adenovirus vectors. Inall cases, after infusing the dextrans, the inventors observed the rapidappearance of both dextrans in the kidney by intravital two-photonmicroscopy. Large molecular weight dextran molecules were restricted tothe vasculature, while low molecular weight dextran molecules passed theglomerular filtration barrier, where they gained access to the lumens ofproximal tubules, and were rapidly endocytosed by proximal tubuleepithelial cells, and were then concentrated within the distal tubulelumens (FIG. 8D). Importantly, dextrans were taken up equally well bycells expressing fluorescent proteins, indicating that these cells wereviable and metabolically active. These data were confirmed by histologystudies (FIGS. 8G and 8H), that showed normal renal structure withinthis timeframe. However, baculovirus vectors appeared to alter renalstructure beyond the 3 day period.

D. Serum Creatinine Levels and Vital Signs are Unaffected by theHydrodynamic Transgene Delivery Process

The inventors monitored creatinine levels in normal rats that receivedhydrodynamic injections of saline alone or vectors. Creatinine levels inthese rats remained within normal baseline levels (0.3 to 0.5 mg/dl)throughout the measurement period of up to 14 days after receivinghydrodynamic fluid delivery. There was no significant difference in thelevels in rats that received isotonic fluid and those that receivedvectors. Similarly, blood pressure, body temperature and heart rate wereall unaffected by the injection process.

E. Pressurized Retrograde Venous Injections Provide Widespread Deliveryof Exogenous Macromolecules to the Kidney, and Restricts itsDistribution to the Target Kidney

The inventors attempted to clarify the mechanism that permitted highlyefficient introduction of exogenous genes into the cells of the kidney.The inventors first investigated the extent of renal uptake that couldbe attained with solutions injected using this method. For thesestudies, live rats received hydrodynamic injections of 0.5 ml oftoluidine dye solutions. The inventors then harvested whole left andright kidneys, hearts, livers, lungs and spleens from these rats.Sagittal plane sections of these organs revealed robust distribution ofthe toluidine dye within the left (injected) kidney, and no traceswithin the contralateral kidney and the other organs examined when theinjection process was performed as described above.

In comparison, hydrodynamic injections that were conducted withoutclamping the renal artery and vein (an approach used unsuccessfully inthe early attempts to achieve expression of fluorescent proteins)resulted in minimal uptake of the dye within the target organ (leftkidney), and significant levels within the aforementioned offsite andhighly vascular organs.

F. Hydrodynamic Delivery Facilitates the Robust Cellular Internalizationof Low, Intermediate and High Molecular Weight Exogenous MacromoleculesThroughout Live Kidneys

The inventors next investigated whether hydrodynamic infusions couldreliably facilitate the cellular uptake of large macromolecules invarious nephron segments in live animals. For this study, salinesolutions containing either both low (3 kDa Cascade Blue), andintermediate (Texas Red labeled albumin) or large (150 kDa TRITC) oronly low molecular weight dextrans were injected into the left renalveins of live rats.

The kidneys were imaged within 20 minutes after these fine-needleinjections. In this case the inventors observed widespread distributionof the dextrans in vivo (FIG. 8). Remarkably, this pressurized injectionfacilitated robust and widespread apical and basolateral (FIG. 8)distribution and cellular internalization of albumin, and largemolecular weight TRITC and FITC dextran molecules within tubularepithelial cells in a fashion similar to the incorporation of lowmolecular weight dextran molecules into proximal tubular cells (FIG.8D).

The inventors also observed that albumin and large molecular weightdextran molecules were uncharacteristically able to access the tubulelumen at high concentrations after being delivered to the kidney viahydrodynamic injections (FIG. 8C). Similarly, when 150 kDa molecules,were introduced into the bloodstream prior to hydrodynamic injection ofsaline, they were internalized within tubular epithelial cells.Nevertheless, this atypical access for large molecular weight dextranmolecules to tubule lumens and tubular epithelial cells, was transientand appeared to only occur for molecules present at the time of thehydrodynamic injection process, as 150 kDa dextran molecules infused viathe jugular vein approximately 20-30 minutes after a hydrodynamicpressurized injection of saline remained confined to the vasculature(FIG. 8F).

G. Parameters Related to Renal Transformation

In order to characterize parameters related to effective transformation,the inventors recorded changes in renal venous pressures generatedduring the hydrodynamic injection procedure in the renal vein of liverats. From these measurements, the inventors observed that theapplication and removal of the vascular clamps produced small transientchanges in renal pressure. The hydrodynamic fluid delivery producedpressure responses that generally lasted the duration of the infusions.Overall renal venous pressures increased by up to 25 mmHg (FIG. 15).

This implied that hydrodynamic injections generated significant, yettransient increases in regular renal venous and peritubular capillarypressures.

The inventors next examined the conditions required to inject transgenesat infusion rates lower than that advised for hydrodynamic delivery. Theinventors performed 2- and 4-minute long injections. These comparablylow infusion rate injections increased periods of venous cannulation,and did not produce significant changes in venous pressure.

Interestingly, these lower injections rates also generated successfultransgene expression, see FIG. 14. However, as previously mentioned,4-minute long injections allowed prolonged entry of the 30-guage needleinto the venous cavity. This resulted in extensive bleeding and beyond15 minutes of vessel occlusion to induce hemostasis. According toliterature, this insult is known to produce acute kidney injury, whichis characteristic of the observed in vivo and in vitro tissue damage.These data suggests that lower hydrodynamic infusions rates can generatesignificant renal injury.

Example 22 Acute Kidney Injury Therapy

All renal injuries were generated using micro-serrefines. Rats wereanesthetized from intraperitoneal injections of 50 mg/kg pentobarbital,and then placed on a heating pad to maintain normal physiological bodytemperature. Once fully sedated, their abdomen was shaved, cleaned withbetadine solution and midline incisions were created to isolate therenal pedicles. Thereafter, bilateral renal pedicle clamps were used toocclude blood flow for two specific periods: 10-15 and 30-45 minutes.These damp times correspond to mild, acute kidney injuries respectively.After each period of ischemia, the micro-serrefines were removed toreinstate renal blood flow and the animals were prepared to receivehydrodynamic transgene delivery 60 minutes and 24 hours (timeframe formaximal injury with AKI) after ischemia/reperfusion injury. In the caseof the 24-hour injection time point, each rat was allowed to recoverfrom the effects of the anesthetic. After isolating the renal veins insedated normal and injured rats, the inventors elevating this vein witha silk loop and clamped the renal artery and then the vein. A 0.5 mltransgene solution (transgenes suspended in saline were used todetermine if the inventors could simultaneously induce exogenous proteinexpression in live animals, while providing a therapeutic benefit fromthe fluid injection) or saline was then rapidly injected into the vein,distal to the clamp. Again after this injection, pressure was applied tothe injection site for approximately three minutes. The inventors thenremoved the venous clamp, followed by the arterial clamp, and preparedthe animal for recovery. The inventors collected sera from these animalsacross a period of 72 hours to investigate the changes in creatininethat may be obtained using hydrodynamic fluid delivery. From theresults, the inventors determined that hydrodynamic fluid delivered atthe maximal time of injury (24 hours) returned serum creatinine normallevels in rats with AKI. In comparison, animals with AKI that did notreceive any intervention remained with elevated creatinine levels asanticipated. Moreover, serum creatinine levels in normal rats were notaffected by hydrodynamic delivery, this result suggests that thehydrodynamic fluid delivery process does not appear to have adebilitating affect on overall renal function. Similarly, in rats withmild ischemia there were also no recorded increases in serum creatininevalues, as again anticipated.

Mean Mean Mean Mean Mean Mean Experimental Creatinine CreatinineCreatinine Creatinine Creatinine Creatinine Model Day 0 Day 1 Day 2 Day3 Day 4 Day 5 Normal 0.4 0.4 0.5 0.4 0.4 0.3 AKI 0.47 3.97 3.39 2.85 1.60.6 AKI + HD 1 hour post 0.35 3.9 3.2 1.95 1.1 0.55 injury AKI + HD 24hour post 0.37 2.92 2.4 1.54 0.57 0.5 injury

In addition, the inventors used pressurized retrograde renal veininjections to deliver mitochondrial genes IDH2 and suphotransferase tonormal rats and waited for a period of seven days. Moderateischemia-reperfusion injury was then induced using the bilateral renalclamp model. The serum creatinine levels were monitored before and afterinducing the injury. It was determined that rats that receivedhydrodynamic injections of approximately 600 μg of the plasmids wereresistant to acute kidney injury that was generated by moderate ischemiareperfusion. See FIG. 18.

Example 23 Ischemia Therapy

Ischemia-reperfusion injuries remain a significant clinical problem, asapproximately 25% of ICU patients experience acute kidney injury (AKI).These patients have increased risk of end-stage renal failure, andmortality. Therapy of AKI depends on the identification and treatment ofits underlying cause(s), yet current treatment regimens are mainlysupportive. In the absence of hypervolemia, intravenous fluid deliveryis oftentimes the first course of treatment. This standard approach isemployed to prevent or eliminate volume depletion, ameliorate tubularblockage, dilute nephrotoxin, facilitate diuresis and restore normalGFR. In this study, the inventors investigated the therapeutic potentialof a relatively low volume (0.5 ml) hydrodynamic isotonic fluid deliveryto the left renal vein 1 and 24 hours after inducing moderateischemia-reperfusion injury. Strikingly, from only the fluid deliveredat the 24-hour mark, the inventors observed substantial andstatistically significant (p-value=0.02) decrease in serum creatinine ascompared to control untreated animals. The creatinine levels were alsosignificantly different (p-value=0.03) from those obtained after fluiddelivery at the 1 hour time point. Additionally, hydrodynamic fluiddelivery provided at the 24 hour mark mediated a return to baselineserum creatinine levels within 4 days of the initial insult. Thepotential therapeutic benefit observed in these results provides anexciting platform to facilitate the future management ofischemia-reperfusion injuries using in a single infusion technique.

Renal injury was generated using renal pedicle cross clamps. Rats wereanesthetized from intraperitoneal injections of 50 mg/kg pentobarbital,and then placed on a heating pad to maintain normal physiologicaltemperature. Using a standard model to generate renal injury, bilateralrenal pedicle clamps were applied to occlude blood flow for periods of10-15 and 30-45 minutes. These clamp period correspond to mild andmoderate/acute kidney injuries respectively. At the end of each period,the clamps were removed to reinstate renal blood flow and the animalswere prepared to receive hydrodynamic transgene delivery at either 1 or24 hours after ischemia/reperfusion injury (the 24 hour time pointcorresponds to the period of maximal damage in AKI). After isolating theleft renal vein in each sedated rat, the inventors elevated the veinwith a 4-0 silk loop, and clamped the renal artery and then the vein.The left kidney was chosen over the right vein primarily because it iseasier to conduct the necessary surgical manipulations on this site inthe mammal. A 0.5 ml transgene solution was then rapidly injected intothe vein, distal to the clamp. Pressure was then applied to theinjection site for approximately three minutes to induce hemostasis. Theinventors then removed the venous clamp, followed by the arterial clamp,and prepared the animal for recovery.

Prior to attempting hydrodynamic transgene delivery in rats with anyform of renal ischemia/reperfusion injury, the inventors firstdetermined whether it was possible to use this technique to successfullydeliver exogenous substances to injured kidneys. To answer thisquestion, the inventors compared the results obtained from thehydrodynamic delivery of fluorescent dextrans in injured kidneys to thatin normal kidneys. Intravital micrographs, data presented in FIG. 11,were taken from both groups of rats, within 20 minutes of them receivinghydrodynamic infusions of 0.5 ml saline containing 4 kDa FITC (lowmolecular weight) and 150 kDa TRITC signals (large molecular weight)dextrans, and 30 ul of Hoechst 33342. The Hoechst 33342 was added toidentify cellular nuclei. FIG. 11A illustrates the distribution of thehydrodynamically delivered probes in normal rat kidney. Intense TRITCsignals are confined to the vasculature, and FITC conjugated dextransdelineate brush borders of proximal tubules and are observed asinternalized puncta within tubular epithelial cells. Moreover, the FITCdye appears more concentrated within the lumen of the distal tubules.These observations are consistent with previously presented data thatoutline intact structural and functional renal capacities.

Using intravital fluorescent multiphoton, microscopy micrographs werethen acquired from live rats that received hydrodynamic transgeneinjections at the time points 1 and 24 hours after inducing mild andacute ischemia/reperfusion injuries. In these micrographs, FIG. 13,transgene-expressed GFP fluorescence is observed within proximal tubuleepithelial cells and within the lumens of occluded tubules of live ratsthat received plasmid injected treatment at both investigated injectiontime points. The distinctive fluorescent pattern observed along proximaltubule brush borders in normal rats, FIG. 12, was also present in ratswith the mild form of injury, FIG. 13. However, this pattern was absentin rats with moderate ischemia/reperfusion injury, as seen in FIGS. 14and 15. As expected, there was also a substantial disruption to normalrenal architecture in the rats that received the moderate form ofinjury. This made it at times particularly difficult to make morphologicdistinctions between proximal and distal tubules, as shown in FIG. 14D.

Additionally, the inventors estimated the degree of transgene expressionin live renal segments by determining the percentage of renal segments(primarily tubules) within a microscopic field that expressed thetransgenes. A segment was considered to be transfected as long as atleast one of its cells expressed GFP. Thereafter, the inventors averagedthis value across 10 adjacent microscopic fields to provide ourestimate. This estimation provided a 70-90% transfection efficiency ratein superficial cortex that is accessible by intravital multiphotonmicroscopy, in both groups of rats with moderate ischemia/reperfusioninjuries. These estimated efficiencies were greater than those obtainedfor normal rats and rats with a mild form of ischemia/reperfusioninjury, which ranged from approximately 60-70%, FIGS. 16 and 17.

H. The Apparatus for Hydrodynamic Pressure Delivery to Restore RenalFunction.

Referring now to FIG. 27, there is illustrated an end portion of ahydrodynamic pressure delivery catheter, shown generally at 100. Thecatheter 100 includes an insertion end 102 having an injection lumen 104and at least one pressure sensor 106. The injection lumen 104 provides afluid delivery conduit between the supply source (i.e., pump, syringe,etc.) and the target organ (e.g. a kidney). In the illustratedembodiment, the pressure sensor 106 includes two pickups that areelectrically connected, by wires 106 a embedded in the catheter sectionof the embodiment shown in FIGS. 29 and 30, to a control/pumping unit,as will be described below. The pressure sensor 106 may be configuredother than illustrated and remain within the scope of the invention. Thepressure sensor 106 is provided to measure fluid pressure at thecatheter insertion end 102. The fluid pressure may be, for example,systolic/diastolic blood pressure, fluid delivery pressure, and/or thesummed value of delivered fluid pressure moving against the residualblood pressure present during the procedure. Fluid delivery pressure tothe injection lumen 104 may also be measured within the control/pumpunit such that line losses or pressure differentiation with residualblood pressures may be accounted for and fluid delivery pressuresadjusted accordingly.

The insertion end 102 is illustrated having a tip section 108 thatdefines a region having a reduced or minimum diameter, compared to thediameters of other sections of the catheter 100, as will be describedbelow. In a schematic illustration of the catheter 100 shown in FIG. 31,the diameter of the tip section 108 is generally similar to a diameter,D1, of a body portion 101 of the catheter 100, which, in one embodiment,may be in a range of 30 to 40 French. In other embodiments, differentcatheter size ranges may be used, which may be, at least in part, basedon the physical size of the mammal and organs. The tip section 108extends between the forward most point of the insertion end 102 and astabilizer 110. The tip section 108 may be any length desired or may beomitted in certain applications. As shown in FIG. 31, the tip length,L1, may be in a range from about 0.1 cm to about 1.0 cm, with oneembodiment having a length of about 0.7 cm. The stabilizer 110 includesone or more radially extending elements, which are illustrated in FIGS.27, 28A, and 28B as three inflatable balloon sections 112. The balloonsections 112 may be any number of sections and, in other embodiments,there may be more than three sections that are positioned about thecircumference of the catheter 100. The stabilizer 110 is configured togenerally center the insertion end 102, and particularly the end of theinjection lumen 104, within a tissue passage such as a vein or arteryand the like, as shown in FIG. 33. The stabilizer 110 further serves todampen vibratory pulsations that occur in response to the injected fluidpressure and volume, which occur over a generally short timeframe. Thestabilizer 110 substantially reduces oscillations of the insertion end102 to reduce both fluid velocity losses from contact with the venousside walls and tissue damage that may result from the forces at theinsertion end 102. Though illustrated as three separate balloon sections112, in another embodiment, the stabilizer 110 may be configured to havea single balloon section 112 having thinned and thickened sections topermit a multi-legged structure, such as a triangle, star, and the likewhen pressurized by a fluid, such as air, water, saline, and the like.In one embodiment, as shown in FIG. 31, the stabilizer 110 may have adiameter D3 in a range of about 0.5 cm to about 1.5 cm, when freelyexpanded. In another embodiment, the stabilizer 110 may have a diameterD3 in a range of about 0.1 cm to about 0.75 cm, when freely expanded. Inan embodiment of the stabilizer 110, a stabilizer length (L2-L1) may bein a range of 0.8 cm to about 1.0 cm, where the length L2 is in a rangeof about 1.5 to 1.7 cm.

Referring to FIGS. 34-37, several examples of alternative stabilizerconfigurations are illustrated. As shown in FIG. 34, a catheter 200includes a stabilizer 210 configured as a single balloon structureformed in a spiral, screw thread shape. FIGS. 35A, 35B, and 36illustrate an alternative configuration of a multi-legged stabilizer310, formed as part of a catheter 300. The stabilizer 310 is illustratedhaving three balloon segments 312 that are expandable when filled with afluid. It should be understood that any number of balloon segments 312may be provided and remain within the scope of the invention. As shownin FIG. 36, the balloon segments 312 are formed to circumferentiallywrap around a section of the catheter 300 prior to inflation. Wheninflated, the balloon segments 312 rotate and radially expand outwardlytoward the vein wall. Referring to FIG. 37, a cross section of astabilizer 410 includes three balloon segments 412 distributed in anequally spaced arrangement around an injection lumen 404. The balloonsegments 412 include a thickened contact section 414 and thinner wallsection 416. When inflated, the balloon segments 412 expand radiallyoutwardly such that the thickened contact sections 414 bear against thevein wall and the thinner sections 416 circumferentially expand toprovide a tuned vibratory system in response to the fluid dynamicsemanating from the injection lumen 404. The circumferentially expandedwall sections may also provide dampening for fluid/blood pressure andflow moving past the stabilizer 410.

In the illustrated embodiment of FIGS. 27, 29 and 30, the balloonsections 112 are fluidly interconnected by way of a conduit 114 a thatis in fluid communication with a stabilizer actuating lumen 114 b. Thestabilizer actuating lumen 114 b extends between a pump and the conduit114 a, or alternatively a single balloon section 112, to radially expandthe stabilizer 110. The stabilizer 110 is configured to center theinsertion end 102 within the tissue passage yet permit at least aportion of fluid or fluid pressure to be conducted toward an occludingballoon 116. The occluding balloon is expanded by way of an actuatinglumen 116 a that is connected to an external fluid source. The occludingballoon 116 is illustrated as a single inflatable balloon. In analternative embodiment shown in FIG. 28C, an occluding balloon 216 maybe a series of annular balloon rings 216 a that are spaced apart. Wheninflated, the expanded rings 216 a may further capture tissue betweenadjacent rings to assist in securing the catheter during hydrodynamicpressure delivery.

A trap 118 is provided between the stabilizer 110 and the occludingballoon 116, as shown in FIGS. 27 and 28A, though such is not required.The trap 118 is illustrated as a section having a diameter that isgenerally similar to the diameter D1 of a catheter body 101, whichextends between the catheter external source connections the occludingballoon 116. The trap 118 may have a diameter that is similar to thediameter of the insertion end 102. In another embodiment, the trap 118has a diameter that is larger than the diameter, D1 of the catheter body101. Generally, the trap 118 has a diameter that is smaller than theinflated diameters of the stabilizer 112 and the occluding balloon 116.The trap 118 may have a length such that a portion of the tissue passagemay be captured, trapped, or otherwise compressed between the stabilizer110 and the occluding balloon 116. The trap 118 may also be configuredso that the tissue is not compressed or otherwise retained between thestabilizer 112 and the occluding balloon 118, as shown in FIG. 33. Inthe embodiment shown in FIG. 31, the trap 118 has a length (L3-L2) ofabout 0.2 cm to about 0.5 cm, where the dimension L3 is in a range ofabout 1.9 cm to about 2 cm.

Referring now to FIGS. 38 and 39, there is illustrated an embodiment ofa pump, shown generally at 500. The pump 500 includes a fluidcontainment vessel 502, illustrated as a syringe, though any suitablevessel may be used if desired. The syringe 502 is held in place byclamps 504 and includes a plunger 506 connected to an injection controlunit 508, illustrated as an actuator. Alternatively, when other vesseldevices are used, the plunger 506 may be part of the actuator 508. Inone embodiment, the actuator 508 is a displacement-controlled steppermotor that accurately regulates the discharge flow rate of fluidcontained in the syringe 502. The syringe 502 is coupled to theinjection lumen 104 of the catheter 100 by way of an injection coupling510. The actuator 508 is controlled by an algorithm that is part of acontroller 570, which includes an integrated circuit board having one ormore microprocessors. The controller 570 regulates the actuator speedbased on fluid volume in the syringe 502, inputted fluid delivery timeand pressure, and feedback from various sensors that measure injectionparameters. The pump 500 includes a display 512 to communicate status tothe operator. The pump 500 further includes various controls, such asstart, stop, cancel, pause, and programming inputs. The programminginputs may alternatively be provided by a computer that is incommunication with the pump 500.

The control algorithm of the controller 570 may include a pressure/timefluid injection curve, similar to FIG. 32. The curve includes variouspressure points that are either monitoring pressures or set pointpressure parameters. For example P1 may be indicative of a vascularclamp pressure applied event, signaling the initiation of the timesequence of the method, and P3 which may be indicative of a vascularclamp pressure removed event, signaling the cessation or verification ofcessation of occlusion and or stabilization operations. P2 is indicativeof the fluid pressure delivered to the kidney from the injection lumen104 of the insertion end 102, as measured by the pressure sensors 106.P2 may be a set point pressure in the algorithm that is associated witha volume delivered over time calculation. In one embodiment, thealgorithm controls fluid delivery at a rate in a range of about 0.05milliliters/second (ml/sec) to about 0.2 ml/sec, and in a specific fluiddelivery rate of about 0.1 ml/sec. Such a fluid delivery rate issuitable for small mammals, such as for example laboratory mice andrats. In another embodiment, the algorithm controls fluid delivery at arate in a range of about 0.7 ml/sec to about 1.3 ml/sec, and in aspecific fluid delivery rate of about 1 ml/sec. Such a fluid deliveryrate is suitable for larger mammals and humans. In yet anotherembodiment, the fluid delivery rate is in a range of about 0.25 ml/secto about 20 ml/sec. In one embodiment, the volume of fluid that isdelivered to an organ is in a range of about 25 ml to about 250 ml. Inone embodiment, a total fluid volume of about 60 ml is delivered into ahuman kidney. In one embodiment, the algorithm further controls thefluid delivery pressure such that the pressure P2 is maintained in arange of about 25 mm Hg to about 35 mm Hg, with a specific targetpressure P2 of about 30 mm Hg. In another embodiment, the algorithm maycontrol the fluid delivery pressure within a range of about 5 mm Hg toabout 140 mm Hg. The algorithm may, alternatively or in conjunction witha pre-programmed fluid pressure range, provide an efficacious pressuredelta increase over a predetermined time is in a range of about 100% to1,000% over a baseline pressure. In one embodiment, the ratio of kidneyorgan size to P2 pressure is a generally linear relationship betweenvarious mammal sizes. Thus, the rat/mouse pressure, P2 and kidney sizeis a first point and the human pressure, P2 and kidney size is a secondpoint, permitting a linear extrapolation for animal sizes therebetween.

An occlusion pump 520 is in fluidic communication with the occludingballoon 116 by way of the actuating lumen 116 a coupled to an occludingcoupling 522. The occluding pump 520 is also operated according to analgorithm in the controller 570. The controller 570 may be configured toregulate the rate of inflation and the occlusion balloon pressure, basedin part on the type of fluid used. Similarly, a stabilizer pump 530 isin fluidic communication with the stabilizer 110 by way of thestabilizer actuating lumen 114 b coupled to a stabilizer coupling 532.The stabilizer pump 530 is also operated according to an algorithm inthe controller 570. The occlusion and stabilizer pumps may be furtherregulated by the controller 570 based on feedback from various sensorsthat measure inflation parameters, such as fluid pressure, blood flow,pulse rate and the like. The controller 570 may be a single controlleror controllers coupled to individual subsystems, if desired. Thepressure sensor 106 is coupled to the controller 570 by way of aconnector 540 to provide tip pressure inputs to the algorithm.

Referring now to FIGS. 42 and 43, there is shown another example of apressurization protocol embodiment that is compatible with the algorithmdescribed above. The embodiment of FIG. 42 shows a variation of thevarious pressure stages, P1, P2, and P3, of FIG. 32. These pressurestages have been expanded into finer incremental sub-steps or stages andmay include higher pressure levels. Associated with each step in theaccompanying table of the example of FIG. 42 is a target venous pressureand an expected venous pressure, as measured by a pressure sensor, suchas is described above in conjunction with either the catheter or thepump. For this example, the pressure data shown may be interpreted asrelative values to those of Steps A and I. Thus, absolute values may befractional values or whole multiplier values of those expressed in FIG.42, including a whole multiplier of 1.0. As shown, Step A, identified as“steady state pressure,” represents the time period prior to theinitiation of Pressure P1 of FIG. 32. In this specific embodiment, thesteady state pressure of the example curve is shown in a range of about10-12 mm Hg. Steps B and C are identified as “beginning of ballooninflation” and “balloon fully inflated,” respectively, and correspond tothe vascular clamp pressure applied event P1. In the illustratedembodiment, the pressure at Step B elevates in a generally linearramping function, though such is not required. The pressure increase maybe measured within the vein and is related to the increase in bloodpressure within the vein between the occluding balloons and/orstabilizers and the downstream blockage or restriction within an organ.As shown in FIG. 42, the resulting venous pressure is indicated as about40 mm Hg.

Referring next to Steps D, E, and F, these steps correspond to the stageof fluid delivery pressure identified as P2. In this example, Step Dillustrates an injection pressure increase to an activation pressure ofStep E. The injection pressure of Step D is shown as a two-leg pressureprofile where the pressurized injection of fluid is ramped at a firstrate, shown as from about 40 to about 42 mm Hg. A second rate increasesthe pressure to about 60 mm Hg, which is the activation pressure of StepE. In one embodiment, the rate of the pressure rise is characterized asan impulse pressure rise that occurs over a short and abrupt time frame.In the illustrated embodiment, the time frame may be 5 seconds or lessand may be up to about 15 seconds. Once achieved, the activationpressure is held at a generally constant level for the duration of theinjection cycle. During this time a therapeutic volume of fluid isinjected into the organ, as described above. The duration of thepressure holding time may be varied to permit injection of the entiretherapeutic volume, such as 60 cc's of fluid, or may be held for a timeperiod only such that a blockage (i.e., clot, injured tissue, or otherobstacle to flow) or collapsed portion within the pressurized organ isovercome or re-expanded. Referring now to FIG. 43, there is shown a datatable of applied flow rates and the resultant target pressures achieved,as measured in a pig renal vein over the depicted tests.

A Method of Using the Hydrodynamic Pressure Delivery Apparatus.

Referring now to FIGS. 33, 40 and 41, there are illustrated varioussteps in a method of hydrodynamic fluid pressure delivery to an organ,such as a kidney. A syringe 502, filled with fluid, including any of thefluid media disclosed herein, is loaded into the pump 500. The physicianor administering technician programs the controller 570 with theparameters to input a pressure/time curve, similar to that of FIG. 32.The pressure/time curve is a function of the type and size ofpatient/subject/specimen and the particular kidney affliction ortreatment regimen desired.

As shown in FIG. 40, an incision is made, for example, to access theright or left renal vein, by way of the inferior vena cava. Thehydrodynamic pressure delivery catheter 100 is routed into the renalvein by conventional means, such as guide wires and other knownmanipulation devices. As shown in FIGS. 33 and 41, the catheter isdirected towards the kidney and inserted far enough into the renal veinto permit occlusion and isolation of the organ. In one example, thecatheter and, in particular the occluding balloon, is inserted past thegonadal vessels (testicular vein) so that pressurized fluid is preventedfrom entering the gonadal vessels.

Once inserted into position, as shown in FIG. 41, the occluding balloon116 may be inflated first to close off blood flow from the kidney. Thestabilizer 110 may be inflated concomitant with, prior to, or afterinflation of the occluding balloon. The stabilizer 110, in addition to aprimary function of stabilizing and centering the catheter tip, may alsoprovide an anchoring function, in consort with the occluding balloon116, to maintain the position of the catheter 100 in the renal vein. Asshown in FIG. 33, the occluding balloon 116 is inflated and expandsfirmly against the vein inner wall to seal off blood flow exiting thekidney. The stabilizer 110 is inflated and contacts the vein inner wallat several points around the inner diameter thereof. The stabilizer 110pilots, or otherwise generally centers the end of the injection lumen104 in the vein. During the pressurization event, the stabilizerradially secures the catheter insertion end 102 and dampens oscillationsassociated with rapid fluid flow through the injection lumen 104. In oneembodiment, the inflation sequence and the amount of fluid used toinflate the occluding balloon 116 and the stabilizer 110 are controlledby the respective occluding pump 520 and stabilizer pump 530. The pumps520 and 530 may further include a pressure sensor and a feedback loop topermit a predetermined pressure (and the related fluid volume) to bemaintained during the pressurization procedure.

Once the catheter 100 is positioned and secured in place, thepressurization sequence may be initiated. When the sequence starts, theactuator 508 drives the syringe plunger to expel the fluid at theprogrammed delivery rate. Fluid is ducted through the injection lumen104 against the pressurized blood volume at the insertion end 102. Thefluid is driven at a relatively high rate, compared to current kidneytreatment regimens, and enters the kidney. As illustrated in FIG. 41,the fluid volume cause an expansion of the kidney. While not wishing tobe bound by theory, this expansion appears to cause a stretch activatedresponse throughout the kidney. The mechanical expansion of the kidneytissues and nephrons are believed to drive the fluid past the capillarystructures within the kidney and facilitating cellular uptake of thefluid.

The principle and mode of operation of this invention have beenexplained and illustrated in its preferred embodiment. However, it mustbe understood that this invention may be practiced otherwise than asspecifically explained and illustrated without departing from its spiritor scope.

1.-26. (canceled)
 27. A catheter for delivering a fluid to a subject viaa lumen of a blood vessel comprising: a body portion with an injectionlumen extending longitudinally through the body portion to an insertionend of the catheter, and a stabilizer on an external surface of thecatheter configured to at least partially prevent movement of thecatheter within the lumen of the blood vessel and/or to inhibit bloodflow within the blood vessel.
 28. The catheter of claim 27, wherein thestabilizer is a fluid inflatable stabilizer comprising one or moreradially extendable and contractible balloons sized to be expanded intocontact with the lumen of the blood vessel, and the catheter furthercomprises a stabilizer actuating lumen in fluid communication with thefluid inflatable stabilizer for conducting an inflation fluid to andfrom the fluid inflatable stabilizer.
 29. The catheter of claim 28,wherein the one or more radially extendable and contractible balloons isof a type selected from the group consisting of: a radially extendingballoon having thinned and thickened sections configured to extendradially as a multi-legged structure when inflated; a radially expandingballoon wound spirally around the body portion; and one or more radiallyextending balloons having variable thickness to provide a contactsection which has a greater thickness than a wall section
 30. Thecatheter of claim 27, further comprising a fluid inflatable occludingballoon positioned around the body portion further from the insertionend than the fluid inflatable stabilizer, and an occluding balloonactuating lumen in fluid communication with the occluding balloon forconducting an inflation fluid to and from the fluid inflatable occludingballoon.
 31. The catheter of claim 27, further comprising at least onepressure sensor and a control unit, wherein the at least one pressuresensor is in communication with the control unit, and the at least onepressure sensor is configured to measure at least one of blood pressure,fluid delivery pressure, and internal kidney pressure and provide afeedback signal to the control unit.
 32. A system for delivery of afluid to a kidney of a subject via a lumen of a renal blood vesselcomprising: a catheter adapted for insertion into the renal blood vesselcomprising: a body portion with an injection lumen extendinglongitudinally through the body portion to an insertion end of thecatheter, and a stabilizer on an external surface of the catheterconfigured to at least partially prevent movement of the catheter withinthe lumen and/or to inhibit blood flow within the blood vessel; at leastone fluid containment vessel, wherein at least one fluid containmentvessel is in fluid communication with the injection lumen; at least onecontrol unit; at least one pump or actuator controllable by the leastone control unit, wherein the at least one pump or actuator is operableto force a fluid from the at least one fluid containment vessel throughthe injection lumen; and at least one pressure sensor in communicationwith the at least one control unit, wherein the at least one pressuresensor is configured to measure at least one of blood pressure, fluiddelivery pressure, and internal kidney pressure.
 33. The system of claim32, wherein the stabilizer is a fluid inflatable stabilizer comprisingone or more radially extendable and contractible balloons sized to beexpanded into contact with the lumen of the blood vessel, and thecatheter further comprises a stabilizer actuating lumen in fluidcommunication with the fluid inflatable stabilizer for conducting aninflation fluid to and from the fluid inflatable stabilizer.
 34. Thesystem of claim 32, wherein the catheter further comprises a fluidinflatable occluding balloon positioned around the body portion furtherfrom the insertion end than the stabilizer and an occluding balloonactuating lumen in fluid communication with the fluid inflatableoccluding balloon for conducting an inflation fluid to and from thefluid inflatable occluding balloon.
 35. The system of claim 32, whereinthe at least one pressure sensor delivers at least one of bloodpressure, fluid delivery pressure, and internal kidney pressuremeasurements to the at least one control unit, wherein the at least onecontrol unit comprises a control algorithm configured to control adelivery pressure of the fluid through the injection lumen by regulatinga pump or actuator in functional communication with a fluid containmentvessel comprising the fluid.
 36. The system of claim 35, wherein the atleast one control unit is programmable with a pressure/time curve andthe control unit regulates the delivery pressure of the fluid throughthe injection lumen over time according to the pressure/time curve. 37.The system of claim 32, wherein the stabilizer actuating lumen is influid communication with a fluid containment vessel comprising aninflation fluid and the at least one control unit comprises a controlalgorithm configured to control delivery of the inflation fluid to thefluid inflatable stabilizer by regulating a pump or actuator infunctional communication with the fluid containment vessel to maintainthe insertion end in an approximately centered position within the lumenof the renal blood vessel in response to the measurements delivered tothe at least one control unit by the at least one pressure sensor. 38.The system of claim 37, wherein the catheter further comprises a fluidinflatable occluding balloon positioned around the body portion furtherfrom the insertion end than the stabilizer and an occluding balloonactuating lumen in fluid communication with the fluid inflatableoccluding balloon for conducting an inflation fluid to and from thefluid inflatable occluding balloon, wherein the occluding balloonactuating lumen is in fluid communication with a fluid containmentvessel comprising an inflation fluid and the at least one control unitcomprises a control algorithm configured to control delivery of theinflation fluid to the fluid inflatable occluding balloon by regulatinga pump or actuator in functional communication with the fluidcontainment vessel to maintain occlusion of the lumen of the renal bloodvessel in response to the measurements delivered to the at least onecontrol unit by the at least one pressure sensor.
 39. The system ofclaim 32, wherein the system maintains the delivery pressure over a timeperiod sufficient to cause uptake of the fluid into at least one cell ofthe kidney or to clear debris from the kidney.
 40. The system of claim32, wherein the system is configured to realize at least one of:providing a delivery pressure of about 5 mm Hg to about 140 mm Hg; andcausing a pressure delta increase of about 100% to about 1,000% overtime relative to a baseline pressure.
 41. A method for delivering afluid to a kidney of a subject via a lumen of a renal vein comprising:introducing into the lumen of the renal blood vessel a cathetercomprising a body portion with an injection lumen extendinglongitudinally through the body portion to an insertion end of thecatheter; stabilizing the insertion end within the lumen of the renalvein; occluding the renal vein; measuring at least one of bloodpressure, fluid delivery pressure, and internal kidney pressure; anddelivering the fluid through the injection lumen to the kidney at a flowrate sufficient to maintain a preselected internal kidney pressure overa preselected period of time.
 42. The method of claim 50, wherein avolume of fluid of about 25 ml to about 250 ml is delivered to thekidney at a flow rate sufficient to maintain a hydrodynamic pressure inthe kidney of about 5 mm Hg to about 140 mm Hg for a period of time, orat a flow rate sufficient to cause a pressure delta increase over abaseline pressure in the kidney of about 100% to about 1,000% over aperiod of time.
 43. The method of claim 41, wherein the insertion end isstabilized within the lumen of the renal vein by inflating a fluidinflatable stabilizer comprising one or more radially extendable andcontractible balloons positioned rearward from the insertion end, andthe renal blood vessel is occluded by at least one of the fluidinflatable stabilizer or an inflated fluid inflatable occluding balloonpositioned further from the insertion end than the fluid inflatablestabilizer relative.
 44. The method of claim 41, wherein the fluidcomprises at least one element selected from the group of: nucleicacids; adenovirus vectors; stem cells; renal epithelial cells;fibroblasts; endothelial cells; plasmids; artificial chromosomes;retroviruses; adenovirus; adeno-associated virus; anti-sense DNA; siRNA;ShRNA; RNAi; organelles-mitochondria; peroxisomes; endosomes; exosomes;hormones; growth factors; peptides; derivatized peptides and proteins;glycosylated proteins; non-glycosylated proteins; sugar; polymers;drugs; saline; lactated ringers; saline with glucose; and bicarbonate.45. A method for ameliorating symptoms of, or treating acute kidneyinjury in a subject in need thereof, comprising performing the method ofclaim
 41. 46. The method of claim 60, wherein the method of claim 45 isperformed from about 1 hour to about 24 hours following onset of acutekidney injury in the subject.